Plant cytokinin oxidase

ABSTRACT

The present invention relates to methods for stimulating root growth and/or enhancing the formation of lateral or adventitious roots and/or altering root geotropism comprising expression of a plant cytokinin oxidase or comprising expression of another protein that reduces the level of active cytokinins in plants or plant parts. The invention also relates to novel plant cytokinin oxidase proteins, nucleic acid sequences encoding cytokinin oxidase proteins as well as to vectors, host cells, transgenic cells and plants comprising said sequences. The invention also relates to the use of said sequences for improving root-related characteristics including increasing yield and/or enhancing early vigor and/or modifying root/shoot ratio and/or improving resistance to lodging and/or increasing drought tolerance and/or promoting in vitro propagation of explants and/or modifying cell fate and/or plant development and/or plant morphology and/or plant biochemistry and/or plant physiology. The invention also relates to the use of said sequences in the above-mentioned methods. The invention also relates to methods for identifying and obtaining proteins and compounds interacting with cytokinin oxidase proteins. The invention also relates to the use of said compounds as a plant growth regulator or herbicide.

FIELD OF THE INVENTION

The present invention generally relates to a method for modifying plantmorphological, biochemical and physiological properties orcharacteristics, such as one or more developmental processes and/orenvironmental adaptive processes, including but not limited to themodification of initiation or stimulation or enhancement of root growth,and/or adventitious root formation, and/or lateral root formation,and/or root geotropism, and/or shoot growth, and/or apical dominance,and/or branching, and/or timing of senescence, and/or timing offlowering, and/or flower formation, and/or seed development, and/or seedyield, said method comprising expressing a cytokinin degradation controlprotein, in particular cytokinin oxidase, in the plant, operably underthe control of a regulatable promoter sequence such as a cell-specificpromoter, tissue-specific promoter, or organ-specific promoter sequence.Preferably, the characteristics modified by the present invention arecytokinin-mediated and/or auxin-mediated characteristics. The presentinvention extends to genetic constructs which are useful for performingthe inventive method and to transgenic plants produced therewith havingaltered morphological and/or biochemical and/or physiological propertiescompared to their otherwise isogenic counterparts.

BACKGROUND OF THE INVENTION

Roots are an important organ of higher plants. Their main functions areanchoring of the plant in the soil and uptake of water and nutrients(N-nutrition, minerals, etc.). Thus, root growth has a direct orindirect influence on growth and yield of aerial organs, particularlyunder conditions of nutrient limitation. Roots are also relevant for theproduction of secondary plant products, such as defense compounds andplant hormones.

Roots are also storage organs in a number of important staple crops.Sugar beet is the most important plant for sugar production in Europe(260 Mill t/year; 38% of world production). Manioc (cassaya), yams andsweet potato (batate) are important starch producers (app. 150 Millt/year each). Their content in starch can be twice as high as that ofpotato. Roots are also the relevant organ for consumption in a number ofvegetables (e.g. carrots, radish), herbs (e.g. ginger, kukuma) andmedicinal plants (e.g. ginseng). In addition, some of the secondaryplant products found in roots are of economic importance for thechemical and pharmaceutical industry. An example is yams, which containbasic molecules for the synthesis of steroid hormones. Another exampleis shikonin, which is produced by the roots of Lithospermumerythrorhizon in hairy root cultures. Shikonin is used for itsanti-inflammatory, anti-tumour and wound-healing properties.

Moreover, improved root growth of crop plants will also enhancecompetitiveness with weedy plants and will improve growth in arid areas,by increasing water accessibility and uptake.

Improved root growth is also relevant for ecological purposes, such asbioremediation and prevention/arrest of soil erosion.

Root architecture is an area that has remained largely unexploredthrough classical breeding, because of difficulties with assessing thistrait in the field. Thus, biotechnology could have significant impact onthe improvement of this trait, because it does not rely on large-scalescreenings in the field. Rather, biotechnological approaches require abasic understanding of the molecular components that determine aspecific characteristic of the plant. Today, this knowledge is onlyfragmentary, and as a consequence, biotechnology was so far unable torealize a break-through in this area.

A well-established regulator of root growth is auxin. Application ofindole-3-acetic acid (IAA) to growing plants stimulates lateral rootdevelopment and lateral root elongation (Torrey, Am J Bot 37: 257–264,1950; Blakely et al., Bot Gaz 143: 341–352, 1982; Muday and Haworth,Plant Physiol Biochem 32: 193–203, 1994). Roots exposed to a range ofconcentrations of IAA initiated increasing numbers of lateral roots(Kerk et al., Plant Physiol, 122: 925–932, 2000). Furthermore, whenroots that had produced laterals in response to a particularconcentration of exogenous auxin were subsequently exposed to a higherconcentration of IAA, numerous supernumerary lateral roots spacedbetween existing ones were formed (Kerk et al., Plant Physiol, 122:925–932, 2000). Conversely, growth of roots on agar containingauxin-transport inhibitors, including NPA, decreases the number oflateral roots (Muday and Haworth, Plant Physiol Biochem 32: 193–203,1994).

Arabidopsis mutants containing Increased levels of endogenous IAA havebeen isolated (Boerjan et al., Plant Cell 7: 1405–141, 1995; Celenza etal., Gene Dev 9: 2131–2142, 1995; King et al., Plant Cell 7: 2023–2037,1995; Lehman et al., Cell 85: 183–194, 1996). They are now known to bealleles of a single locus located on chromosome 2. These mutantseedlings have excess adventitious and lateral roots, which is inaccordance with the above-described effects of external auxinapplication.

The stimulatory effect of auxins on adventitious and lateral rootformation suggests that overproduction of auxins in transgenic plants isa valid strategy for increasing root growth. Yet, it is alsoquestionable whether this would yield a commercial product with improvedcharacteristics. Apart from its stimulatory effect on adventitious andlateral root formation, auxin overproduction triggers other effects,such as reduction in leaf number, abnormal leaf morphology (narrow,curled leaves), aborted inflorescences, increased apical dominance,adventitios root formation on the stem, most of which are undesirablefrom an agronomic perspective (Klee et al., Genes Devel 1: 8696, 1987;Kares et al., Plant Mol Biol 15: 225–236, 1990). Therefore, the majorproblem with approaches that rely on increased auxin synthesis is aproblem of containment, namely to confine the effects of auxin to theroot. This problem of containment is not likely overcome by usingtissue-specific promoters: auxins are transported in the plant and theiraction is consequently not confined to the site of synthesis. Anotherissue is whether auxins will always enhance the total root biomass. Foragar-grown plants, it has been noticed that increasing concentrationsprogressively stimulated lateral root formation but concurrentlyinhibited the outgrowth of these roots (Kerk et al., Plant Physiol, 122:925932, 2000).

The above-mentioned problems related to containment of auxin effects andto maintenance of root outgrowth are solved by the embodiments in thepatent claim.

SUMMARY OF THE INVENTION

The present invention relates to a genetic construct comprising a geneencoding a protein with cytokinin oxidase activity from Arabidopsisthaliana. This gene is expressed under control of a regulated promoter.This promoter may be regulated by endogenous tissue-specific orenvironment-specific factors or, alternatively, it may be induced byapplication of specific chemicals.

The present invention also relates to a cell or plant containing thegenetic construct.

The present invention also relates to a method to modify rootarchitecture and biomass by expression of a cytokinin oxidase gene undercontrol of a promoter that is specific to the root or to certain tissuesor cell types of the root.

DETAILED DESCRIPTION OF THE INVENTION

To by-pass above-mentioned problems associated with increasing auxinbiosynthesis, it was decided to follow an alternative approach. Wereasoned that down-regulation of biological antagonists of auxins couldevoke similar or even superior effects on root growth as compared toincreasing auxin levels. Hormone actions and interactions are extremelycomplex, but we hypothesized that cytokinins could function as auxinantagonists with respect to root growth. Hormone studies on plant tissuecultures have shown that the ratio of auxin versus cytokinin is moreimportant for organogenesis than the absolute levels of each of thesehormones, which indeed indicates that these hormones function asantagonists—at least in certain biological processes. Furthermore,lateral root formation is inhibited by exogenous application ofcytokinins. Interestingly, also root elongation is negatively affectedby cytokinin treatment, which suggests that cytokinins control both rootbranching and root outgrowth.

Together, current literature data indicate that increasing cytokininlevels negatively affects root growth, but the mechanisms underlyingthis process are not understood. The sites of cytokinin synthesis in theplant are root tips and young tissues of the shoot. Endogenousconcentrations of cytokinins are in the nM range. However, as theirquantification is difficult, rather large tissue amounts need to beextracted and actual local concentrations are not known. Also thesubcellular compartmentation of cytokinins is not known. It is generallythought that the free base and ribosides are localized in the cytoplasmand nucleus, while glucosides are localized in the vacuole. There existalso different cytokinins with slightly different chemical structure. Asa consequence, it is not known whether the effects of exogenouscytokinins should be ascribed to a raise in total cytokininconcentration or rather to the competing out of other forms ofplant-borne cytokinins (which differ either in structure, cellular orsubcellular location) for receptors, translocators, transporters,modifying enzymes . . .

In order to test the hypothesis that cytokinin levels in the root indeedexceed the level optimal for root growth, novel genes encoding cytokininoxidases (which are cytokinin metabolizing enzymes) were cloned fromArabidopsis thaliana (designated AtCKX) and were subsequently expressedunder a strong constitutive promoter in transgenic tobacco andArabidopsis. Transformants showing AtCKX mRNA expression and increasedcytokinin oxidase activity also manifested enhanced formation and growthof roots.

Negative effects on shoot growth were also observed. The latter is inaccordance with the constitutive expression of the cytokinin oxidasegene in these plants, illustrating the importance of confined expressionof the cytokinin oxidase gene for general plant growth properties.Containment of cytokinin oxidase actMty can be achieved by using cell-,tissue- or organ-specific promoters, since cytokinin degradation is aprocess limited to the tissues or cells that express the CKX protein,this in contrast to approaches relying on hormone synthesis, asexplained above.

The observed negative effects of cytokinin oxidase expression on shootgrowth demonstrate that cytokinin oxidases are interesting targets forthe design of or screening for growth-promoting chemicals. Suchchemicals should inhibit cytokinin oxidase activity, should preferablynot be transported to the root and should be rapidly degraded in soil,so that application of these chemicals will not inhibit root growth.Cytokinins also delay leaf senescence, which means that positive effectswill include both growth and maintenance of photosynthetic tissues. Inaddition, the observation that cytokinins delay senescence, enhancegreening (chlorophyll content) of leaves and reduce shoot apicaldominance shows that strategies based on suppressing CKX activity (suchas antisense, ribozyme, and cosuppression technology) in the aerialparts of the plant could result in delayed senescence, enhanced leafgreening and increased branching.

Similarly, the observed positive effects of cytokinin oxidase expressionon root growth demonstrate that cytokinin oxidases are interestingtargets for the design of or screening for herbicides. Such herbicidesshould inhibit cytokinin oxidase activity, should preferably not betransported to the shoot, and should be soluble and relatively stable ina solvent that can be administered to the root through the soil.

These effects of cytokinin oxidase overexpression on plant developmentand architecture were hitherto unknown and, as a consequence, thepresented invention and its embodiments could not be envisaged.

The observed negative effects on shoot growth demonstrate thatmanipulation of cytokinin oxidases can also be used for obtainingdwarfing phenotypes. Dwarfing phenotypes are particularly useful incommercial crops such as cereals and fruit trees for example.

Preferable embodiments of the invention relate to the positive effect ofcytokinin oxidase expression on plant growth and architecture, and inparticular on root growth and architecture. The cytokinin oxidase genefamily contains at least six members in Arabidopsis (see examples below)and the present inventors have shown that there are quantitativedifferences in the effects achieved with some of these genes intransgenic plants. It is anticipated that functional homologs of thedescribed Arabidopsis cytokinin oxidases can be isolated from otherorganisms, given the evidence for the presence of cytokinin oxidaseactivity in many green plants (Hare and van Staden, Physiol Plant91:128–136, 1994; Jones and Schreiber, Plant Growth Reg 23:123–134,1997), as well as in other organisms (Armstrong, in Cytokinins:Chemistry, Activity and Function. Eds Mok and Mok, CRC Press, pp139–154,1994). Therefore, the sequence of the cytokinin oxidase, functional inthe invention, need not to be identical to those described herein. Thisinvention is particularly useful for cereal crops and monocot crops ingeneral and cytokinin oxidase genes from for example wheat or maize maybe used as well (Morris et al., 1999; Rinaldi and Comandini, 1999). Itis envisaged that other genes with cytokinin oxidase activity or withany other cytokinin metabolizing activity (see Za{hacek over (z)}ímalováet al., Biochemistry and Molecular Biology of Plant Hormones, Hooykaas,Hall and Libbenga (Eds.), Elsevier Science, pp141–160, 1997) can also beused for the purpose of this invention. Similarly, genes encodingproteins that would increase endogenous cytokinin metabolizing activitycan also be used for the purpose of this invention. In principle,similar phenotypes could also be obtained by interfering with genes thatfunction downstream of cytokinin such as receptors or proteins involvedin signal transduction pathways of cytokinin.

For the purpose of this invention, it should be understood that the term‘root growth’ encompasses all aspects of growth of the different partsthat make up the root system at different stages of its development,both in monocotyledonous and dicotyledonous plants. It is to beunderstood that enhanced growth of the root can result from enhancedgrowth of one or more of its parts including the primary root, lateralroots, adventitious roots, etc. all of which fall within the scope ofthis invention.

According to a first embodiment, the present invention relates to amethod for stimulating root growth and/or enhancing the formation oflateral and/or adventitious roots and/or altering root geotropismcomprising expression of a plant cytokinin oxidase or comprisingexpression of another protein that reduces the level of activecytokinins in plants or plant parts.

In the context of the present invention it should be understood that theterm “expression” and/or ‘overexpression’ are used interchangeably andboth relate to an “enhanced and/or ectopic expression” of a plantcytokinin oxidase or any other protein that reduces the level of activecytokinins in plants. It should be clear that herewith an enhancedexpression of the plant cytokinin oxidase as well as “de novo”expression of plant cytokinin oxidases or of said other proteins ismeant. Alternatively, said other protein enhances the cytokininmetabolizing activity of a plant cytokinin oxidase.

It futher should be understood that in the context of the presentinvention the expression “lateral and/or adventitious roots” can mean“lateral and adventitious roots” but also “lateral or adventitiousroots”. The enhancement can exist in the formation of lateral roots orin the formation of adventitious roots as well as in the formation ofboth types of non-primary roots, but not necessarily.

According to a further embodiment, the present invention relates to amethod for stimulating root growth and/or enhancing the formation oflateral or adventitious roots and/or altering root geotropism and/orincreasing yield and/or enhancing early vigor and/or modifyingroot/shoot ratio and/or improving resistance to lodging and/orincreasing drought tolerance and/or promoting in vitro propagation ofexplants, comprising expression of a plant cytokinin oxidase orcomprising expression of another protein that reduces the level ofactive cytokinins in plants or plant parts.

According to a preferred embodiment, the present invention relates to amethod for stimulating root growth resulting in an increase of root massby overexpression of a cytokinin oxidase, preferably a cytokinin oxidaseaccording to the invention, or another protein that reduces the level ofactive cytokinins in plants or plant parts, preferably in roots.

Higher root biomass production due to overexpression of growth promotingsequences has a direct effect on the yield and an indirect effect ofproduction of compounds produced by root cells or transgenic root cellsor cell cultures of said transgenic root cells. One example of aninteresting compound produced in root cultures is shikonin, the yield ofwhich can be advantageously enhanced by said methods.

According to a more specific embodiment, the present invention relatesto a method for stimulating root growth or for enhancing the formationof lateral and adventitious roots or for altering root geotropismcomprising expression of a nucleic acid encoding a plant cytokininoxidase selected from the group consisting of:

-   -   (a) nucleic acids comprising a DNA sequence as given in any of        SEQ ID NOs 27, 1, 3, 5, 7, 9, 11, 25, 26, 28 to 31, 33 or 34, or        the complement thereof,    -   (b) nucleic acids comprising the RNA sequences corresponding to        any of SEQ ID NOs 27, 1, 3, 5, 7, 9, 11, 25, 26, 28 to 31, 33 or        34, or the complement thereof,    -   (c) nucleic acids specifically hybridizing to any of SEQ ID NOs        27, 1, 3, 5, 7, 9, 11, 25, 26, 28 to 31, 33 or 34, or to the        complement thereof,    -   (d) nucleic acids encoding a protein comprising the amino acid        sequence as given in any of SEQ ID NOs 2, 4, 6, 8, 10, 12, 32 or        35, or the complement thereof,    -   (e) nucleic acids as defined in any of (a) to (d) characterized        in that said nucleic acid is DNA, genomic DNA, cDNA, synthetic        DNA or RNA wherein T is replaced by U,    -   (f) nucleic acids which are degenerated to a nucleic acid as        given in any of SEQ ID NOs 27, 1, 3, 5, 7, 9, 11, 25, 26, 28 to        31, 33 or 34, or which are degenerated to a nucleic acid as        defined in any of (a) to (e) as a result of the genetic code,    -   (g) nucleic acids which are diverging from a nucleic acid        encoding a protein as given in any of SEQ ID NOs 2, 4, 6, 8, 10,        12 or 35 or which are diverging from a nucleic acid as defined        in any of (a) to (e), due to the differences in codon usage        between the organisms,    -   (h) nucleic acids encoding a protein as given in SEQ ID NOs 2,        4, 6, 8, 10, 12 or 35 or nucleic acids as defined in (a) to (e)        which are diverging due to the differences between alleles,    -   (i) nucleic acids encoding a protein as given in any of SEQ ID        NOs 2, 4, 6, 8, 10, 12 or 35,    -   (j) functional fragments of nucleic acids as defined in any        of (a) to (i) having the biological activity of a cytokinin        oxidase, and    -   (k) nucleic acids encoding a plant cytokinin oxidase,        or comprising expression, preferably in roots, of a nucleic acid        encoding a protein that reduces the level of active cytokinins        in plants or plant parts.

In the present invention, nucleic acids encoding novel Arabidopsisthaliana cytokinine oxidases have been isolated and for the first time,the present inventors suprisingly could show that the expression ofcytokinin oxidases in transgenic plants or in transgenic plant partsresulted in the above-mentioned root-related features. Preferably, theexpression of the cytokinine oxidase(s) should take place in roots,preferably under the control of a root-specific promoter. One example ofsuch a root-specific promoter is provided in SEQ ID NO 36.

It should be clear that, although the invention is supported in theexamples section by several new AtCKX genes and proteins, the inventiveconcept also relates to the use of other cytokinin oxidases isolatedfrom and expressed in other plants, preferably in the roots of saidother plants to obtain similar effects in plants as desribed in theexamples section.

Therefore, the present invention more generally relates to the use of anucleic acid encoding a plant cytokinin oxidase or encoding a proteinthat reduces the level of active cytokinins in plants or plant parts forstimulating root growth or for enhancing the formation of lateral oradventitious roots or for altering root geotropism. Preferred cytokininoxidases to be used are encoded by the nucleic acids encoding thecytokinin oxidases as defined above and are encoded by the novel nucleicacids of the invention as defined hereunder.

The invention relates to an isolated nucleic acid encoding a novel plantprotein having cytokinin oxidase activity selected from the groupconsisting of:

-   -   (a) a nucleic acid comprising a DNA sequence as given in any of        SEQ ID NOs 29, 3, 5, 9, 26, 27, 31, 33 or 34, or the complement        thereof,    -   (b) a nucleic acid comprising the RNA sequences corresponding to        any of. SEQ ID NOs 29, 3, 5, 9, 26, 27, 31, 33 or 34, or the        complement thereof,    -   (c) a nucleic acid specifically hybridizing to a nucleic acid as        given in any of SEQ ID NOs 29, 3, 5, 9, 26, 27, 31, 33 or 34, or        the complement thereof,    -   (d) a nucleic acid encoding a protein with an amino acid        sequence comprising the polypeptide as given in SEQ ID NO 32 and        which is at least 70% similar, preferably at least 75%, 80% or        85%, more preferably at least 90% or 95%, most preferably at        least 99% similar to the amino acid sequence as given in SEQ ID        NO 4,    -   (e) a nucleic acid encoding a protein with an amino acid        sequence which is at (east 35% similar, preferably 37%, 40%,        45%, 47% or 50%, similar, more preferably 55%, 60%, 65%, 70%,        75% or 80% similar, most preferably 85%, 90% or 95% similar to        the amino acid sequence as given in SEQ ID NO 6,    -   (f) a nucleic acid encoding a protein with an amino acid        sequence which is at least 35% similar, preferably 37%, 40%,        45%, 47% or 50%, similar, more preferably 55%, 60%, 65%, 70%,        75% or 80% similar, most preferably 85%, 90% or 95% similar to        the amino acid sequence as given in SEQ ID NO 10 or 35,    -   (g) a nucleic acid encoding a protein comprising the amino acid        sequence as given in any of SEQ ID NOs 4, 6, 10, 32 or 35,    -   (h) a nucleic acid which is degenerated to a nucleic acid as        given in any of SEQ ID NOs 29, 3, 5, 9, 26, 27, 33 or 34 or        which is degenerated to a nucleic acid as defined in any of (a)        to (g) as a result of the genetic code,    -   (i) a nucleic acid which is diverging from a nucleic acid        encoding a protein as given in any of SEQ ID NOs 4, 6, 10 or 35        or which is diverging from a nucleic acid as defined in any        of (a) to (g) due to the differences in codon usage between the        organisms,    -   (j) a nucleic acid encoding a protein as given in SEQ ID NOs 4,        6, 10 or 35, or a nucleic acid as defined in (a) to (g) which is        diverging due to the differences between alleles,    -   (k) a nucleic acid encoding an immunologically active fragment        of a cytokinin oxidase encoded by a nucleic acid as given in any        of SEQ ID NOs 29, 3, 5, 9, 26, 27, 31, 33 or 34, or an        immunologically active fragment of a nucleic acid as defined in        any of (a) to (j),    -   (l) a nucleic acid encoding a functional fragment of a cytokinin        oxidase encoded by a nucleic acid as given in any of SEQ ID NOs        29, 3, 5, 9, 26, 27, 31, 33 or 34, or a functional fragment of a        nucleic acid as defined in any of (a) to (j), wherein said        fragment has the biological activity of a cytokinin oxidase, and    -   (m) a nucleic acid encoding a protein as defined in SEQ ID NO 4,        6, 10 or 35,        provided that said nucleic acid is not the nucleic acid as        deposited under any of the following Genbank accession numbers:        AC005917, AB024035, and AC023754

The invention also relates to an isolated nucleic acid of the inventionwhich is DNA, cDNA, genomic DNA or synthetic DNA, or RNA wherein T isreplaced by U.

The invention also relates to a nucleic acid molecule of at least 15nucleotides in length hybridizing specifically with or specificallyamplifying a nucleic acid of the invention. According to anotherembodiment, the invention also relates to a vector comprising a nucleicacid of the invention. In a preferred embodiment, said vector is anexpression vector wherein the nucleic acid is operably linked to one ormore control sequences allowing the expression of said sequence inprokaryotic and/or eukaryotic host cells.

It should be understood that for expression of the cytokinin oxidasegenes of the invention in monocots, a nucleic acid sequencecorresponding to the cDNA sequence should be used to avoid mis-splicingof introns in monocots. Preferred cDNA sequences to be expressed inmonocots have a nucleic acid sequence as represented in any of SEQ IDNOs 25 to 30 and 34.

The invention also relates to a host cell containing any of the nucleicacid molecules or vectors of the invention. Said host cell is chosenfrom the group comprising bacterial, insect, fungal, plant or animalcells.

Another embodiment of the invention relates to an isolated polypeptideencodable by a nucleic acid of the invention, or a homologue or aderivative thereof, or an immunologically active or a functionalfragment thereof. Preferred polypeptides of the invention comprise theamino acid sequences as represented in any of SEQ ID NOs 2, 4, 6, 8, 10,12, 32 and 35, or a homologue or a derivative thereof, or animmunologically active and/or functional fragment thereof. In an evenmore preferred embodiment, the invention relates to a polypeptide whichhas an amino acid sequence as given in SEQ ID NO 2, 4, 6, 8, 10, 12 or35, or a homologue or a derivative thereof, or an immunologically activeand/or functional fragment thereof. Preferred functional fragmentsthereof are those fragments which are devoid of their signal peptide.

According to yet another embodiment, the invention relates to a methodfor producing a polypeptide of the invention comprising culturing a hostcell of the invention under conditions allowing the expression of thepolypeptide and recovering the produced polypeptide from the culture.

The invention also relates to an antibody specifically recognizing apolypeptide of the invention or a specific epitope thereof.

The invention further relates to a method for the production oftransgenic plants, plant cells or plant tissues comprising theintroduction of a nucleic acid molecule of the invention in anexpressible format or a vector of the invention in said plant, plantcell or plant tissue.

The invention also relates to a method for the production of alteredplants, plant cells or plant tissues comprising the introduction of apolypeptide of the invention directly into a cell, a tissue or an organof said plant.

According to another embodiment, the invention relates to a method foreffecting the expression of a polypeptide of the invention comprisingthe introduction of a nucleic acid molecule of the invention operablylinked to one or more control sequences or a vector of the inventionstably into the genome of a plant cell. The invention further relates tothe method as described above further comprising regenerating a plantfrom said plant cell. The invention also relates to a transgenic plantcell comprising a nucleic acid sequence of the invention which isoperably linked to regulatory elements allowing transcription and/orexpression of said nucleic acid in plant cells or obtainable by a methodas explained above.

According to another preferred embodiment, the invention relates to atransgenic plant cell as described here above wherein the nucleic acidof the invention is stably integrated into the genome of said plantcell.

The invention further relates to a transgenic plant or plant tissuecomprising plant cells as herein described and also to a harvestablepart of said transgenic plant, preferably selected from the groupconsisting of seeds, leaves, fruits, stem cultures, roots, tubers,rhizomes and bulbs. The invention also relates to the progeny derivedfrom any of said transgenic plants or plant parts.

According to another embodiment, the invention relates to a method forstimulating root growth comprising expression of a nucleic acid of theinvention or comprising expression of another protein that reduces thelevel of active cytokinins in plants or plant parts.

A plant cell or tissue culture is an artificially produced culture ofplants cells or plant tissues that is grown in a special medium, eitherliquid or solid, which provides these plant cells or tissues with allrequirements necessary for growth and/or production of certaincompounds. Plant cell and/or tissue cultures can be used for the rapidpropagation of plants and for the production of transgenic plant to namea few examples. Root formation can be difficult for some explants orunder some conditions in said cultures and expression of a cytokininoxidase gene in said cultured plant cells or tissue(s) can be used toenhance root formation. Plant cell and/or tissue culture can also beused for the industrial production of valuable compounds. Possibleproduction compounds are pharmaceuticals, pesticides, pigments,cosmetics, perfumes, food additives, etc. An example of such a productis shikonin, which is produced by the roots of the plant Lithospermumerythrorhizon. An example of a plant tissue culture is a hairy rootculture, which is an artificially produced mass of hairy roots. Roots ofL. erythrorhizon are difficult to collect in large numbers and bypreparing hairy root cultures, the end product shikonin could beindustrially prepared at a faster rate than would normally occur. Asdisclosed herein, expression of cytokinin oxidases enhances root growthand development and can therefore be used advantageously in said plantcell and tissue culture procedures. Therefore, according to anotherembodiment of this invention, a method is provided for stimulating rootgrowth and development comprising expression of a nucleic acid encodinga plant cytokinin oxidase, preferably a cytokinin oxidase of theinvention, in a transgenic plant cell or tissue culture comprising saidtransgenic plant cells.

The invention further relates to a method for enhancing the formation oflateral or adventitious roots comprising expression of a nucleic acid ofthe invention or comprising expression of another protein that reducesthe level of active cytokinins in plants or plant parts.

The invention also relates to method for altering root geotropismcomprising altering the expression of a nucleic acid of the invention orcomprising expression of another protein that that reduces the level ofactive cytokinins in plants or plant parts.

The invention also relates to methods for enhancing early vigor and/orfor modifying is root/shoot ratio and/or for improving resistance tolodging and/or for increasing drought tolerance and/or for promoting invitro propagation of explants comprising expression of a nucleic acid ofthe invention comprising expression of another protein that reduces thelevel of active cytokinins in plants or plant parts.

The invention further relates to methods for increasing the root size orthe size of the root meristem comprising expression of a nucleic acid ofthe invention or comprising expression of another protein that reducesthe level of active cytokinins in plants or plant parts, preferably inroots.

According to yet another embodiment, the invention relates to a methodfor increasing the size of the shoot meristem comprising downregulationof expression of a nucleic acid of the invention, preferably in shoots.

According to a preferred embodiment the invention relates to a methodfor delaying leaf senescence comprising downregulation of expression ofany of the cytokinin oxidases of the invention in leaves, preferably insenescing leaves. Also the invention relates to a method for alteringleaf senescence comprising expression of one of the cytokinin oxidasesin senescing leaves.

The invention also relates to methods for increasing leaf thicknesscomprising expression of a nucleic acid of the invention or comprisingexpression of another protein that reduces the level of activecytokinins in plants or plant parts, preferably in leaves.

The invention also relates to a method for reducing the vessel sizecomprising expression of a nucleic acid of the invention or comprisingexpression of another protein that reduces the level of activecytokinins in plants or plant parts, preferably in vessels. Theinvention further relates to a method for increasing the vessel sizecomprising downregulation of expression of a nucleic acid of theinvention in plants or plant parts. According to another embodiment, theinvention relates to a method for improving standability of seedlingscomprising expression of a nucleic acid of the invention or comprisingexpression of another protein that reduces the level of activecytokinins in seedlings.

Furthermore, the invention relates to any of the above describedmethods, said method leading to an increase in yield.

The invention further relates to any of the methods of the inventionwherein said expression of said nucleic acid occurs under the control ofa strong constitutive promoter. In a preferred embodiment the inventionrelates to any of the methods of the invention wherein said expressionof said nucleic acid occurs under the control of a promoter that ispreferentially expressed in roots. In Table 5 a non-exhaustive list ofroot specific promoters is included. A preferred promoter to be used inthe methods of the invention is the root clavata homolog promoter,having a sequence as given in SEQ ID NO 36.

According to yet another embodiment, the invention relates to a methodfor modifying cell fate and/or modifying plant development and/ormodifying plant morphology and/or modifying plant biochemistry and/ormodifying plant physiology and/or modifying the cell cycle progressionrate comprising the modification of expression in particular cells,tissues or organs of a plant, of a nucleic acid of the invention.

The invention also relates to a method for obtaining enhanced growth,and/or increased yield and/or altered senescence of a plant cell, tissueand/or organ and/or increased frequence of formation of lateral organsin a plant, comprising the ectopic expression of a nucleic acid of theinvention.

The invention also relates to a method for promoting and extending celldivision activity in cells in adverse growth conditions and/or instress, comprising the ectopic expression of a nucleic acid sequence ofthe invention,

According to yet another embodiment, the invention relates to a methodfor identifying and obtaining proteins interacting with a polypeptide ofthe invention comprising a screening assay wherein a polypeptide of theinvention is used.

In a more preferred embodiment, the invention relates to a method foridentifying and obtaining proteins interacting with a polypeptide of theinvention comprising a two-hybrid screening assay wherein a polypeptideof the invention as a bait and a cDNA library as prey are used.

The invention further relates to a method for modulating the interactionbetween a polypeptide of the invention and interacting protein partnersobtainable by a method as described above.

In a further embodiment, the invention relates to a method foridentifying and obtaining compounds interacting with a polypeptide ofthe invention comprising the steps of:

-   -   a) providing a two-hybrid system wherein a polypeptide of the        invention and an interacting protein partner obtainable by a        method as described above,    -   b) interacting said compound with the complex formed by the        expressed polypeptides as defined in a), and,    -   c) performing (real-time) measurement of interaction of said        compound with said polypeptide or the complex formed by the        expressed polypeptides as defined in a).

The invention further relates to a method for identifying compounds ormixtures of compounds which specifically bind to a polypeptide of theinvention, comprising:

-   -   a) combining a polypeptide of the invention with said compound        or mixtures of compounds under conditions suitable to allow        complex formation, and,    -   b) detecting complex formation, wherein the presence of a        complex identifies a compound or mixture which specifically        binds said polypeptide.

The invention also relates to a method as described above wherein saidcompound or mixture inhibits the activity of said polypeptide of theinvention and can be used for the rational design of chemicals.

According to another embodiment, the invention relates to the use of acompound or mixture identified by means of a method as described aboveas a plant growth regulator or herbicide.

The invention also relates to a method for production of a plant growthregulator or herbicide composition comprising the steps of the compoundscreening methods described above and formulating the compounds obtainedfrom said steps in a suitable form for the application in agriculture orplant cell or tissue culture.

The invention also relates to a method for increasing branchingcomprising expression of a nucleic acid of the invention in plants orplant parts, preferably in stems or axillary buds.

The invention also relates to a method for improving lodging resistancecomprising expression of a nucleic acid of the invention in plants orplant parts, preferably in stems or axillary buds.

The invention also relates to a method for the design of or screeningfor growth-promoting chemicals or herbicides comprising the use of anucleic acid of the invention or a vector of the invention.

According to another embodiment, the invention relates to the use of anucleic acid molecule of the invention, a vector of of the invention ora polypeptide of the invention for increasing yield.

The invention also relates to the use of a nucleic acid molecule of ofthe invention, a vector of the invention or a polypeptide of theinvention for stimulating root growth.

The invention also relates to the use of a nucleic acid molecule of theinvention, a vector of the invention or a polypeptide of the inventionfor enhancing the formation of lateral or adventitious roots.

The invention also relates to the use of a nucleic acid molecule of theinvention, a vector of of the invention or a polypeptide of theinvention for altering root geotropism.

The invention further relates to the use of a nucleic acid molecule ofof the invention, a vector of the invention or a polypeptide of theinvention for enhancing early vigor and/or for modifying root/shootratio and/or for improving resistance to lodging and/or for increasingdrought tolerance and/or for promoting in vitro propagation of explants.

The invention also relates to the use of a nucleic acid molecule of theinvention, a recombinant vector of the invention or a polypeptide of theinvention for modifying plant development and/or for modifying plantmorphology and/or for modifying plant biochemistry and/or for modifyingplant physiology.

According to yet another embodiment, the invention relates to adiagnostic composition comprising at least a nucleic acid molecule ofthe invention, a vector of the invention, a polypeptide of the inventionor an antibody of the invention.

Another embodiment of the current invention relates to the use of atransgenic rootstock that has an enhanced root growth and developmentdue to expression of a cytokinin oxidase in grafting procedures with ascion to produce a plant or tree with improved agricultural orhorticultural characteristics. The scion may be transgenic ornon-transgenic. Specific characteristics envisaged by this embodimentare those conferred by root systems and include improved anchoring ofthe plant/tree in the soil and/or improved uptake of water resulting forexample in improved drought tolerance, and/or improved nutrient uptakefrom the soil and/or improved transport of organic substances throughoutthe plant and/or enhanced secretion of substances into the soil such asfor example phytosiderophores, and/or improved respiration and/orimproved disease resistance and/or enhanced yield. An advantage of usingAtCKX transformed rootstocks for grafting, in addition to their enhancedroot system, is the delayed senescence of leaves on the graft, asdisclosed herein (see FIG. 12A). Preferred plants or trees for thisparticular embodiment include plants or trees that do not grow well ontheir own roots and are grafted in cultivated settings such ascommercially profitable varieties of grapevines, citrus, apricot,almond, plum, peach, apple, pear, cherry, walnut, fig, hazel and loquat.

As mentioned supra, auxins and cytokinins act as antagonists in certainbiological processes. For example, the cytokinin/auxin ratio regulatesthe production of roots and shoots with a high concentration of auxinresulting in organized roots and a high concentration of cytokininsresulting in shoot production. As disclosed in this invention,expression of cytokinin oxidases in tobacco and Arabidopsis results inenhanced root development consistent with enhanced auxin effects. Auxinsare also involved in the development of fruit. Treatment of femaleflower parts with auxin results in the development of parthenocarpicfruit in some plant species. Parthenocarpic fruit development has beengenetically engineered in several horticultural crop plants throughincreased biosynthesis of auxins in the female reproductive organs(WO0105985). Therefore, according to another embodiment, this inventionrelates to a method for inducing the parthenocarpic trait in plants,said method consisting of downregulating the expression of one or morecytokinin oxidases or of another protein that reduces the level ofactive cytokinins in plants or plant parts, preferably in the femalereproductive organs such as the placenta, ovules and tissues derivedtherefrom. The DefH9 promoter region from Antirrhinum majus or one ofits homologues, which confer high expression specificity in placenta andovules, can be used for this purpose.

DEFINITIONS AND ELABORATIONS TO THE EMBODIMENTS

Those skilled in the art will be aware that the invention describedherein is subject to variations and modifications other than thosespecifically described. It is to be understood that the inventiondescribed herein includes all such variations and modifications. Theinvention also includes all such steps, features, compositions andcompounds referred to or indicated in this specification, individuallyor collectively, and any and all combinations of any or more of saidsteps or features.

The present invention is applicable to any plant, in particular amonocotyiedonous plants and dicotyledonous plants including a fodder orforage legume, ornamental plant, food crop, tree, or shrub selected fromthe list comprising Acacia spp., Acer spp., Actinidia spp., Aesculusspp., Agathis australis, Albizia amara, Alsophila tricolor, Andropogonspp., Arachis spp, Areca catechu, Astelia fragrans, Astragalus cicer,Baikiaea plurijuga, Betula spp., Brassica spp., Bruguiera gymnorrhiza,Burkea africana, Butea frondosa, Cadaba farinosa, Calliandra spp,Camellia sinensis, Canna indica, Capsicum spp., Cassia spp., Centroemapubescens, Chaenomeles spp., Cinnamomum cassia, Coffea arabica,Colophospermum mopane, Coronillia varia, Cotoneaster serotina, Crataegusspp., Cucumis spp., Cupressus spp., Cyathea dealbata, Cydonia oblonga,Cryptomeria japonica, Cymbopogon spp., Cynthea dealbata, Cydoniaoblonga, Dalbergia monetaria, Davallia divaricata, Desmodium spp.,Dicksonia squarosa, Diheteropogon amplectens, Dioclea spp, Dolichosspp., Dorycnium rectum, Echinochloa pyramidalis, Ehrartia spp., Eleusinecoracana, Eragrestis spp., Erythrina spp., Eucalyptus spp., Eucleaschimperi, Eulalia villosa, Fagopyrum spp., Feijoa sellowiana, Fragariaspp., Flemingia spp, Freycinetia banksii, Geranium thunbergii, Ginkgobiloba, Glycine javanica, Gliricidia spp, Gossypium hirsutum, Grevilleaspp., Guibourtia coleosperma, Hedysarum spp., Hemarthia altissima,Heteropogon contortus, Hordeum vulgare, Hyparrhenia rufa, Hypericumerectum, Hyperthelia dissoluta, Indigo incarnata, Iris spp., Leptarrhenapyrolifolia, Lespediza spp., Lettuca spp., Leucaena leucocephala,Loudetia simplex, Lotonus bainesli, Lotus spp., Macrotyloma axillare,Malus spp., Manihot esculenta, Medicago sativa, Metasequolaglyptostroboides, Musa sapientum, Nicotianum spp., Onobrychis spp.,Ornithopus spp., Oryza spp., Peltophorum africanum, Pennisetum spp.,Persea gratissima, Petunia spp., Phaseolus spp., Phoenix canariensis,Phormium cookianum, Photinia spp., Picea glauca, Pinus spp., Pisumsativum, Podocarpus totara, Pogonarthia fleckii, Pogonarthria squarrosa,Populus spp., Prosopis cineraria, Pseudotsuga menziesli, Pterolobiumstellatum, Pyrus communis, Quercus spp., Rhaphiolepsis umbellata,Rhopalostylis sapida, Rhus natalensis, Ribes grossularia, Ribes spp.,Robinia pseudoacacia, Rosa spp., Rubus spp., Salix spp., Schyzachyriumsanguineum, Sciadopitys verticillata, Sequoia sempervirens,Sequoiadendron giganteum, Sorghum bicolor, Spinacia spp., Sporobolusfimbriatus, Stiburus alopecuroides, Stylosanthos humilis, Tadehagi spp,Taxodium distichum, Themeda triandra, Trifolium spp., Triticum spp.,Tsuga heterophylla, Vaccinium spp., Vicia spp. Vitis vinifera, Watsoniapyramidata, Zantedeschia aethiopica, Zea mays, amaranth, artichoke,asparagus, broccoli, brussel sprout, cabbage, canola, carrot,cauliflower, celery, collard greens, flax, kale, lentil, oilseed rape,okra, onion, potato, rice, soybean, straw, sugarbeet, sugar cane,sunflower, tomato, squash, and tea, amongst others, or the seeds of anyplant specifically named above or a issue, cell or organ culture of anyof the above species.

Throughout this specification, unless the context requires otherwise theword “comprise”, and variations such as “comprises” and “comprising”,will be understood to imply the inclusion of a stated integer or step orgroup of integers or steps but not the exclusion of any other integer orstep or group of integers or steps.

As used herein, the term “derived from” shall be taken to Indicate thata particular integer or group of integers has originated from thespecies specified, but has not necessarily been obtained directly fromthe specified source.

The terms “protein(s)”, “peptide(s)” or “oligopeptide(s)”, when usedherein refer to amino acids in a polymeric form of any length. Saidterms also include known amino acid modifications such as disulphidebond formation, cysteinylation, oxidation, glutathionylation,methylation, acetylation, farnesylation, biotinylation, stearoylation,formylation, lipoic acid addition, phosphorylation, sulphation,ubiquitination, myristoylation, palmitoylation, geranylgeranylation,cyclization (e.g. pyroglutamic acid formation), oxidation, deamidation,dehydration, glycosylation (e.g. pentoses, hexosamines,N-acetylhexosamines, deoxyhexoses, hexoses, sialic acid etc.) andacylation as well as non-naturally occurring amino acid residues,L-amino acid residues and D-amino acid residues.

“Homologues” of a protein of the invention are those peptides,oligopeptides, polypeptides, proteins and enzymes which contain aminoacid substitutions, deletions and/or additions relative to the saidprotein with respect to which they are a homologue, without altering oneor more of its functional properties, in particular without reducing theactivity of the resulting. For example, a homologue of said protein willconsist of a bioactive amino acid sequence variant of said protein. Toproduce such homologues, amino acids present in the said protein can bereplaced by other amino acids having similar properties, for examplehydrophobicity, hydrophilicity, hydrophobic moment, antigenicity,propensity to form or break α-helical structures or β-sheet structures,and so on. An overview of physical and chemical properties of aminoacids is given in Table 1. Substitutional variants of a protein of theinvention are those in which at least one residue in said protein aminoacid sequence has been removed and a different residue inserted in itsplace. Amino acid substitutions are typically of single residues, butmay be clustered depending upon functional constraints placed upon thepolypeptide; insertions will usually be of the order of about 1–10 aminoacid residues, and deletions will range from about 1–20 residues.Preferably, amino acid substitutions will comprise conservative aminoacid substitutions, such as those described supra.

TABLE 1 Properties of naturally occurring amino acids. Chargeproperties/ hydrophobicity Side group Amino Acid nonpolar Aliphatic ala,ile, leu, val hydrophobic aliphatic, S-containing met aromatic phe, trpimino pro polar uncharged Aliphatic gly amide asn, gln aromatic tyrhydroxyl ser, thr sulfhydryl cys positively charged Basic arg, his, lysnegatively charged Acidic asp, glu

Insertional amino acid sequence variants of a protein of the inventionare those in which one or more amino acid residues are introduced into apredetermined site in said protein. Insertions can compriseamino-terminal and/or carboxy-terminal fusions as well as intra-sequenceinsertions of single or multiple amino acids. Generally, insertionswithin the amino acid sequence will be smaller than amino or carboxylterminal fusions, of the order of about 1 to 10 residues. Examples ofamino- or carboxy-terminal fusion proteins or peptides include thebinding domain or activation domain of a transcriptional activator asused in a two-hybrid system, phage coat proteins, (histidine)₆-tag,glutathione S-transferase, protein A, maltose-binding protein,dihydrofolate reductase, Tag 100• epitope (EETARFQPGYRS) SEQ ID NO:37,c-myc epitope (EQKLISEEDL) SEQ ID NO:38, FLAG®-epitope (DYKDDDK) SEQ IDNO:39, lacZ, CMP (calmodulin-binding peptide), HA epitope (YPYDVPDYA)SEQ ID NO:40, protein C epitope (EDOVDPRLIDGK) SEQ ID NO:41, and VSVepitope (YTDIEMNRLGK) SEQ ID NO:42.

Deletional variants of a protein of the invention are characterised bythe removal of one or more amino acids from the amino acid sequence ofsaid protein.

Amino acid variants of a protein of the invention may readily be madeusing peptide synthetic techniques well known in the art, such as solidphase peptide synthesis and the like, or by recombinant DNAmanipulations. The manipulation of DNA sequences to produce variantproteins which manifest as substitutional, insertional or deletionalvariants are well known in the art. For example, techniques for makingsubstitution mutations at predetermined sites in DNA having knownsequence are well known to those skilled in the art, such as by M13mutagenesis, T7-Gen in vitro mutagenesis kit (USB, Cleveland, Ohio),QuickChange Site Directed mutagenesis kit (Stratagene, San Diego,Calif.), PCR-mediated site-directed mutagenesis or other site-directedmutagenesis protocols.

In the current invention “identity” and/or “similarity” percentagesbetween DNA sequences and/or proteins are calculated using computerprograms known in the art such as the DNAstar/MegAlign programs incombination with the Clustal method. “Derivatives” of a protein of theinvention are those peptides, oligopeptides, polypeptides, proteins andenzymes which comprise at least about five contiguous amino acidresidues of said polypeptide but which retain the biological activity ofsaid protein. A “derivative” may further comprise additionalnaturally-occurring, altered glycosylated, acylated or non-naturallyoccurring amino acid residues compared to the amino acid sequence of anaturally-occurring form of said polypeptide. Alternatively or inaddition, a derivative may comprise one or more non-amino acidsubstituents compared to the amino acid sequence of anaturally-occurring form of said polypeptide, for example a reportermolecule or other ligand, covalently or non-covalently bound to theamino acid sequence such as, for example, a reporter molecule which isbound thereto to facilitate its detection.

With “immunologically active” is meant that a molecule or specificfragments thereof such as specific epitopes or haptens are recognizedby, i.e. bind to antibodies. Specific epitopes may be determined using,for example, peptide scanning techniques as described in Geysen et al.(1996) (Geysen, H. M., Rodda, S. J. and Mason, T. J. (1986). A prioridelineation of a peptide which mimics a discontinuous antigenicdeterminant. Mol. Immunol. 23, 709–715.).

The term “fragment of a sequence” or “part of a sequence” means atruncated sequence of the original sequence referred to. The truncatedsequence (nucleic acid or protein sequence) can vary widely in length;the minimum size being a sequence of sufficient size to provide asequence with at least a comparable function and/or activity or theoriginal sequence referred to (e.g. “functional fragment”), while themaximum size is not critical. In some applications, the maximum sizeusually is not substantially greater than that required to provide thedesired activity and/or function(s) of the original sequence. Typically,the truncated amino acid sequence will range from about 5 to about 60amino acids in length. More typically, however, the sequence will be amaximum of about 50 amino acids in lenght, preferably a maximum of about60 amino acids. It is usually desirable to select sequences of at leastabout 10, 12 or 15 amino acids, up to a maximum of about 20 or 25 aminoacids.

Functional fragments can also include those comprising an epitope whichis specific for the proteins according to the invention. Preferredfunctional fragments have a length of at least, for example, 5, 10, 25,100, 150 or 200 amino acids.

It should thus be understood that functional fragments can also beimmunologically active fragments or not.

In the context of the current invention are embodied homologues,derivatives and/or immunologically active and/or functional fragments ofthe cytokinin oxidases as defined supra. Particularly preferredhomologues, derivatives and/or immunologically active and/or functionalfragments of the cytokinin oxidase proteins which are contemplated foruse in the current invention are derived from plants, more specificallyfrom Arabidopsis thaliana, even more specifically said cytokininoxidases are the Arabidopsis thaliana (At)CKX, or are capable of beingexpressed therein. The present invention clearly contemplates the use offunctional homologues or derivatives and/or immunologically activefragments of the AtCKX proteins and is not to be limited in applicationto the use of a nucleotide sequence encoding one of said AtCKX proteins.

Any of said proteins, polypeptides, peptides and fragments thereof canbe produced in a biological system, e.g. a cell culture. Alternativelyany of said proteins, polypeptides, peptides and fragments thereof canbe chemically manufactured e.g. by solid phase peptide synthesis. Saidproteins or fragments thereof can be part of a fusion protein as is thecase in e.g. a two-hybrid assay which enables e.g. the identification ofproteins interacting with a cytokinin oxidase according to theinvention.

The proteins or fragments thereof are furthermore useful e.g. tomodulate the interaction between a cytokinin oxidase according to theinvention and interacting protein partners obtained by a method of theinvention. Chemically synthesized peptides are particularly useful e.g.as a source of antigens for the production of antisera and/orantibodies. “Antibodies” include monoclonal, polyclonal, synthetic orheavy chain camel antibodies as well as fragments of antibodies such asFab, Fv or scFv fragments. Monoclonal antibodies can be prepared by thetechniques as described in e.g. Liddle and Cryer (1991) which comprisethe fusion of mouse myeloma cells to spleen cells derived from immunizedanimals. Furthermore, antibodies or fragments thereof to a molecule orfragments thereof can be obtained by using methods as described in e.g.Harlow and Lane (1988). In the case of antibodies directed against smallpeptides such as fragments of a protein of the invention, said peptidesare generally coupled to a carrier protein before immunization ofanimals. Such protein carriers include keyhole limpet hemocyanin (KLH),bovine serum albumin (BSA), ovalbumin and Tetanus toxoid. The carrierprotein enhances the immune response of the animal and provides epitopesfor T-cell receptor binding sites. The term “antibodies” furthermoreincludes derivatives thereof such as labelled antibodies. Antibodylabels include alkaline phosphatase, PKH2, PKH26, PKH67, fluorescein(FITC), Hoechst 33258, R-phycoerythrin (PE), rhodamine (TRITC), QuantumRed, Texas Red, Cy3, biotin, agarose, peroxidase and gold spheres. Toolsin molecular biology relying on antibodies against a protein includeprotein gel blot analysis, screening of expression libraries allowinggene identification, protein quantitative methods including ELISA andRIA, immunoaffinity purification of proteins, immunoprecipitation ofproteins (see e.g. Example 6) and immunolocalization. Other uses ofantibodies and especially of peptide antibodies include the study ofproteolytic processing (Loffler et al. 1994, Woulfe et al. 1994),determination of protein active sites (Lerner 1982), the study ofprecursor and post-translational processing (Baron and Baltimore 1982,Lerner et al. 1981, Sernier et al. 1982), identification of proteindomains involved in protein-protein interactions (Murakami et al. 1992)and the study of exon usage in gene expression (Tamura et al. 1991).

Embodied in the current invention are antibodies specificallyrecognizing a cytokinin oxidase or homologue, derivative or fragmentthereof as defined supra. Preferably said cytokinin oxidase is a plantcytokinin oxidase, more specifically one of the Arabidopsis thalianacytokinin oxidases (AtCKX).

The terms “gene(s)”, “polynucleotide(s)”, “nucleic acid(s)”, “nucleicacid sequence(s)”, “nucleotide sequence(s)”, or “nucleic acidmolecule(s)”, when used herein refer to nucleotides, eitherribonucleotides or deoxyribonucleotides or a combination of both, in apolymeric form of any length. Said terms furthermore includedouble-stranded and single-stranded DNA and RNA. Said terms also includeknown nucleotide modifications such as methylation, cyclization and‘caps’ and substitution of one or more of the naturally occurringnucleotides with an analog such as inosine. Modifications of nucleotidesinclude the addition of acridine, amine, biotin, cascade blue,cholesterol, Cy3®, Cy5®, Cys5.5® Dabcyl, digoxigenin, dinitrophenyl,Edans, 6-FAM, fluorescein, 3′-glyceryl, HEX, IRD-700, IRD-800, JOE,phosphate psoralen, rhodamine, ROX, thiol (SH), spacers, TAMRA, TET,AMCA-S®, SE, BODIPY®, Marina Blue®, Pacific Blue®, Oregon Green®,Rhodamine Green®, Rhodamine Red®, Rhodol Green® and Texas Red®.Polynucleotide backbone modifications include methylphosphonate,2′-OMe-methylphosphonate RNA, phosphorothiorate, RNA, 2′-OMeRNA. Basemodifications include 2-amino-dA, 2-aminopurine, 3′-(ddA), 3′dA(cordycepin), 7-deaza-dA, 8-Br-dA, 8-oxo-dA, N⁶-Me-dA, abasic site(dSpacer), biotin dT, 2′-OMe-5Me-C, 2′-OMe-propynyl-C, 3′-(5Me-dC),3′-(ddC), 5-Br-dC, 5-I-dC, 5-Me-dC, 5-F-dC, carboxy-dT, convertible dA,convertible dC, convertible dG, convertible dT, convertible dU,7-deaza-dG, 8-Br-dG, 8-oxo-dG, O⁶-Me-dG, S6-DNP-dG, 4-methyl-indole,5-nitroindole, 2′-OMe-inosine, 2′-dl, 0⁶-phenyl-dl, 4-methyl-indole,2′-deoxynebularine, 5-nitroindole, 2-aminopurine, dP(purine analogue),dK(pyrimidine analogue), 3-nitropyrrole, 2-thio-dT, 4-thio-dT,biotin-dT, carboxy-dT, O⁴-Me dT, O⁴-triazol dT, 2′-OMe-propynyl-U,5-Br-dU, 2′-dU, 5-F-dU, 5-l-dU, O⁴-triazol dU. Said terms also encompasspeptide nucleic acids (PNAs), a DNA analogue in which the backbone is apseudopeptide consisting of N-(2-aminoethyl)-glycine units rather than asugar. PNAs mimic the behaviour of DNA and bind complementary nucleicacid strands. The neutral backbone of PNA results in stronger bindingand greater specificity than normally achieved. In addition, the uniquechemical, physical and biological properties of PNA have been exploitedto produce powerful blomolecular tools, antisense and antigene agents,molecular probes and biosensors.

The present invention also advantageously provides nucleic acidsequences of at least approximately 15 contiguous nucleotides of anucleic acid according to the invention and preferably from 15 to 50nucleotides. These sequences may, advantageously be used as probes tospecifically hybridise to sequences of the invention as defined above orprimers to initiate specific amplification or replication of sequencesof the invention as defined above, or the like. Such nucleic acidsequences may be produced according to techniques well known in the art,such as by recombinant or synthetic means. They may also be used indiagnostic kits or the like for detecting the presence of a nucleic acidaccording to the invention. These tests generally comprise contactingthe probe with the sample under hybridising conditions and detecting thepresence of any duplex or triplex formation between the probe and anynucleic acid in the sample.

Advantageously, the nucleic acid sequences, according to the inventionmay be produced using such recombinant or synthetic means, such as forexample using PCR cloning mechanisms which generally involve making apair of primers, which may be from approximately 15 to 50 nucleotides toa region of the gene which is desired to be cloned, bringing the primersinto contact with mRNA, cDNA or genomic DNA from a cell, performing apolymerase chain reaction under conditions which bring aboutamplification of the desired region, isolating the amplified region orfragment and recovering the amplified DNA. Generally, such techniques asdefined herein are well known in the art, such as described in Sambrooket al. (Molecular Cloning: a Laboratory Manual, 1989).

A “coding sequence” or “open reading frame” or “ORF” is defined as anucleotide sequence that can be transcribed into mRNA and/or translatedinto a polypeptide when placed under the control of appropriate controlsequences or regulatory sequences, i.e. when said coding sequence or ORFis present in an expressible format. Said coding sequence of ORF isbounded by a 5′ translation start codon and a 3′ translation stop codon.A coding sequence or ORF can include, but is not limited to RNA, mRNA,cDNA, recombinant nucleotide sequences, synthetically manufacturednucleotide sequences or genomic DNA. Said coding sequence or ORF can beinterrupted by intervening nucleic acid sequences.

Genes and coding sequences essentially encoding the same protein butisolated from different sources can consist of substantially divergentnucleic acid sequences. Reciprocally, substantially divergent nucleicacid sequences can be designed to effect expression of essentially thesame protein. Said nucleic acid sequences are the result of e.g. theexistence of different alleles of a given gene, of the degeneracy of thegenetic code or of differences in codon usage. Thus, as indicated inTable 2, amino acids such as methionine and tryptophan are encoded by asingle codon whereas other amino acids such as arginine, leucine andserine can each be translated from up to six different codons.Differences in preferred codon usage are illustrated in Table 3 forAgrobacterium tumefaciens (a bacterium), A. thaliana, M. sativa (twodicotyledonous plants) and Oryza sativa (a monocotyledonous plant). Toextract one example, the codon GGC (for glycine) is the most frequentlyused codon in A. tumefaciens (36.2%0), is the second most frequentlyused codon in O. sativa but is used at much lower frequencies in A.thaliana and M. sativa (9% and 8.4% respectively). Of the four possiblecodons encoding glycine (see Table 2), said GGC codon is most preferablyused in A. tumefaciens and O. sativa. However, in A. thaliana this isthe GGA (and GGU) codon whereas in M. sativa this is the GGU (and GGA)codon.

DNA sequences as defined in the current invention can be interrupted byintervening sequences. With “intervening sequences” is meant any nucleicacid sequence which disrupts a coding sequence comprising said inventiveDNA sequence or which disrupts the expressible format of a DNA sequencecomprising said inventive DNA sequence. Removal of the interveningsequence restores said coding sequence or said expressible format.Examples of intervening sequences include introns and mobilizable DNAsequences such as transposons. With “mobilizable DNA sequence” is meantany DNA sequence that can be mobilized as the result of a recombinationevent.

TABLE 2 Degeneracy of the genetic code. Three- One- letter letter AminoAcid code code Possible codons Alanine Ala A GCA GCC GCG GCU ArginineArg R AGA AGG CGA CGC CGG CGU Asparagine Asn N AAC AAU Aspartic Acid AspD GAC GAU Cysteine Cys C UGC UGU Glutamic Acid Glu E GAA GAG GlutamineGln Q CAA CAG Glycine Gly G GGA GGC GGG GGU Histidine His H CAC CAUIsoleucine Ile I AUA AUC AUU Leucine Leu L UUA UUG CUA CUC CUG CUULysine Lys K AAA AAG Methionine Met M AUG Phenylalanine Phe F UUC UUUProline Pro P CCA CCC CCG CCU Serine Ser S AGC AGU UCA UCC UCG UCUThreonine Thr T ACA ACC ACG ACU Tryptophan Trp W UGG Tyrosine Tyr Y UACUAU Valine Val V GUA GUC GUG GUU Possible “STOP” codons UAA UAG UGA

TABLE 3 Usage of the indicated codons in the different organisms givenas frequency per thousand codons (http://www.kazusa.or.jp/codon).Agrobacterium Arabidopsis Medicago Oryza Codon tumefaciens thalianasativa sativa UUU 13.9 22.5 24.1 11.3 UUC 24.3 20.7 16.9 26.3 UUA 3.512.9 10.4 4.7 UUG 13.2 21.0 22.4 11.8 UCU 7.0 24.6 19.8 10.1 UCC 14.810.8 7.7 16.9 UCA 7.4 17.8 17.2 9.7 UCG 18.2 8.9 3.2 10.8 UAU 12.3 15.216.6 9.2 UAC 10.3 13.7 14.0 20.6 UAA 0.9 0.9 1.2 0.9 UAG 0.6 0.5 0.8 0.8UGU 3.0 10.8 10.6 5.0 UGC 7.4 7.2 5.8 14.3 UGA 1.8 1.0 0.8 1.3 UGG 12.212.7 10.0 12.8 CUU 19.1 24.3 28.3 14.6 CUC 25.7 15.9 12.0 28.0 CUA 5.210.0 8.8 5.7 CUG 31.6 9.9 8.5 22.1 CCU 7.7 18.3 23.2 11.8 CCC 10.6 5.35.3 12.5 CCA 8.9 16.1 22.6 12.2 CCG 20.7 8.3 3.6 16.7 CAU 10.6 14.0 14.69.2 CAC 9.1 8.7 9.1 14.6 CAA 11.2 19.7 23.2 11.9 CAG 24.9 15.2 12.3 24.6CGU 12.2 8.9 10.1 6.8 CGC 25.5 3.7 4.2 15.9 CGA 8.2 6.2 4.2 4.2 CGG 13.24.8 1.8 9.7 AUU 15.4 22.0 29.4 13.8 AUC 36.9 18.5 14.7 25.5 AUA 6.2 12.911.7 7.2 AUG 24.7 24.5 21.7 24.4 ACU 6.4 17.8 20.8 10.3 ACC 20.9 10.311.7 18.6 ACA 9.1 15.9 18.9 10.0 ACG 18.8 7.6 2.8 10.8 AAU 13.5 22.725.0 12.9 AAC 18.7 20.9 18.7 25.1 AAA 13.6 31.0 32.2 12.0 AAG 24.4 32.635.1 39.4 AGU 5.7 14.0 12.6 7.3 AGC 15.8 11.1 8.8 16.9 AGA 5.3 18.7 13.67.7 AGG 6.5 10.9 11.7 14.9 GUU 16.6 27.3 34.7 15.0 GUC 29.3 12.7 9.922.8 GUA 6.1 10.1 10.0 5.7 GUG 19.7 17.5 16.5 25.0 GCU 17.4 28.0 34.619.8 GCC 35.8 10.3 11.4 33.2 GCA 19.5 17.6 25.9 15.6 GCG 31.7 8.8 3.425.3 GAU 25.8 36.8 40.0 21.5 GAC 28.0 17.3 15.5 31.6 GAA 29.9 34.4 35.917.1 GAG 26.3 32.2 27.4 41.1 GGU 16.5 22.2 28.7 16.3 GGC 36.2 9.0 8.434.7 GGA 12.5 23.9 27.3 15.0 GGG 11.3 10.2 7.4 16.6

“Hybridization” is the process wherein substantially homologouscomplementary nucleotide sequences anneal to each other. Thehybridization process can occur entirely in solution, i.e. bothcomplementary nucleic acids are in solution. Tools in molecular biologyrelying on such a process include PCR, subtractive hybridization and DNAsequence determination. The hybridization process can also occur withone of the complementary nucleic acids immobilized to a matrix such asmagnetic beads, Sepharose beads or any other resin. Tools in molecularbiology relying on such a process include the isolation of poly (A+)mRNA. The hybridization process can furthermore occur with one of thecomplementary nucleic acids immobilized to a solid support such as anitrocellulose or nylon membrane or immobilized by e.g. photolitographyto e.g. a silicious glass support (the latter known as nucleic acidarrays or microarrays or as nucleic acid chips). Tools in molecularbiology relying on such a process include RNA and DNA gel blot analysis,colony hybridization, plaque hybridization and microarray hybridization.In order to allow hybridization to occur, the nucleic acid molecules aregenerally thermally or chemically (e.g. by NaOH) denatured to melt adouble strand into two single strands and/or to remove hairpins or othersecondary structures from single stranded nucleic acids. The stringencyof hybridization is influenced by conditions such as temperature, saltconcentration and hybridization buffer composition. High stringencyconditions for hybridization include high temperature and/or low saltconcentration (salts include NaCl and Na3-citrate) and/or the inclusionof formamide in the hybridization buffer and/or lowering theconcentration of compounds such as SDS (detergent) in the hybridizationbuffer and/or exclusion of compounds such as dextran sulfate orpolyethylene glycol (promoting molecular crowding) from thehybridization buffer. Conventional hybridization conditions aredescribed in e.g. Sambrook et al. (1989) but the skilled craftsman willappreciate that numerous different hybridization conditions can bedesigned in function of the known or the expected homology and/or lengthof the nucleic acid sequence. Sufficiently low stringency hybridizationconditions are particularly preferred to isolate nucleic acidsheterologous to the DNA sequences of the invention defined supra.Elements contributing to said heterology include allelism, degenerationof the genetic code and differences in preferred codon usage asdiscussed supra.

Clearly, the current invention embodies the use of the inventive DNAsequences encoding a cytokinin oxidase, homologue, derivative orimmunologically active and/or functional fragment thereof as definedhigher in any method of hybridization. The current invention furthermorealso relates to DNA sequences hybridizing to said inventive DNAsequences. Preferably said cytokinin oxidase is a plant cytokininoxidase, more specifically the Arabidopsis thaliana (At)CKX.

To effect expression of a protein in a cell, tissue or organ, preferablyof plant origin, either the protein may be introduced directly to saidcell, such as by microinjection or ballistic means or alternatively, anisolated nucleic acid molecule encoding said protein may be introducedinto said cell, tissue or organ in an expressible format.

Preferably, the DNA sequence of the invention comprises a codingsequence or open reading frame (ORF) encoding a cytokinin oxidaseprotein or a homologue or derivative thereof or an immunologicallyactive and/or functional fragment thereof as defined supra. Thepreferred protein of the invention comprises the amino acid sequence ofsaid cytokinin oxidase. Preferably said cytokinin oxidase is a plantcytokinin oxidase and more specifically a Arabidopsis thaliana (At)CKX.

With “vector” or “vector sequence” is meant a DNA sequence which can beintroduced in an organism by transformation and can be stably maintainedin said organism. Vector maintenance is possible in e.g. cultures ofEscherichia coli, A. tumefaciens, Saccharomyces cerevisiae orSchizosaccharomyces pombe. Other vectors such as phagemids and cosmidvectors can be maintained and multiplied in bacteria and/or viruses.Vector sequences generally comprise a set of unique sites recognized byrestriction enzymes, the multiple cloning site (MCS), wherein one ormore non-vector sequence(s) can be inserted.

With “non-vector sequence” is accordingly meant a DNA sequence which isintegrated in one or more of the sites of the MCS comprised within avector.

“Expression vectors” form a subset of vectors which, by virtue ofcomprising the appropriate regulatory or control sequences enable thecreation of an expressible format for the inserted non-vectorsequence(s), thus allowing expression of the protein encoded by saidnon-vector sequence(s). Expression vectors are known in the art enablingprotein expression in organisms including bacteria (e.g. E. coli), fungi(e.g. S. cerevisiae, S. pombe, Pichia pastoris), insect cells (e.g.baculoviral expression vectors), animal cells (e.g. COS or CHO cells)and plant cells (e.g. potato virus X-based expression vectors). Thecurrent invention clearly includes any cytokinin oxidase, homologue,derivative and/or immunologically active and/or functional fragmentthereof as defined supra. Preferably said cytokinin oxidase is a plantcytokinin oxidase, more specifically a Arabidopsis thaliana (At)CKX.

As an alternative to expression vector-mediated protein production inbiological systems, chemical protein synthesis can be applied. Syntheticpeptides can be manufactured in solution phase or in solid phase. Solidphase peptide synthesis (Merrifield 1963) is, however, the most commonway and involves the sequential addition of amino acids to create alinear peptide chain. Solid phase peptide synthesis includes cyclesconsisting of three steps: (i) immobilization of the carboxy-terminalamino acid of the growing peptide chain to a solid support or resin;(ii) chain assembly, a process consisting of activation, coupling anddeprotection of the amino acid to be added to the growing peptide chain;and (iii) cleavage involving removal of the completed peptide chain fromthe resin and removal of the protecting groups from the amino acid sidechains. Common approaches in solid phase peptide synthesis includeFmoc/tBu (9-fluorenylmethyloxycarbonyl/t-butyl) and Boc(t-butyloxycarbonyl) as the amino-terminal protecting groups of aminoacids. Amino acid side chain protecting groups include methyl (Me),formyl (CHO), ethyl (Et), acetyl (Ac), t-butyl (t-Bu), anisyl, benzyl(Bzl), trifluroacetyl (Tfa), N-hydroxysuccinimide (ONSu, OSu), benzoyl(Bz), 4-methylbenzyl (Meb), thioanizyl, thiocresyl, benzyloxymethyl(Bom), 4-nitrophenyl (ONp), benzyloxycarbonyl (Z), 2-nitrobenzoyl (NBz),2-nitrophenylsulphenyl (Nps), 4-toluenesulphonyl (Tosyl,Tos),pentafluorophenyl (Pfp), diphenylmethyl (Dpm), 2-chlorobenzyloxycarbonyl(Cl-Z), 2,4,5-trichlorophenyl, 2-bromobenzyloxycarbonyl (Br-Z),tripheylmethyl (Trityl, Trt), and2,5,7,8-pentamethyl-chroman-6-sulphonyl (Pmc). During chain assembly,Fmoc or Boc are removed resulting in an activated amino-terminus of theamino acid residue bound to the growing chain. The carboxy-terminus ofthe incoming amino acid is activated by conversion into a highlyreactive ester, e.g. by HBTU. With current technologies (e.g. PerSeptiveBiosystems 9050 synthesizer, Applied Biosystems Model 431A PeptideSynthesizer), linear peptides of up to 50 residues can be manufactured.A number of guidelines is available to produce peptides that aresuitable for use in biological systems including (i) limiting the use ofdifficult amino acids such as cys, met, trp (easily oxidized and/ordegraded during peptide synthesis) or arg; (ii) minimize hydrophobicamino acids (can impair peptide solubility); and (iii) prevent anamino-terminal glutamic acid (can cyclize to pyroglutamate).

By “expressible format” is meant that the isolated nucleic acid moleculeis in a form suitable for being transcribed into mRNA and/or translatedto produce a protein, either constitutively or following induction by anintracellular or extracellular signal, such as an environmental stimulusor stress (mitogens, anoxia, hypoxia, temperature, salt, light,dehydration, etc) or a chemical compound such as IPTG(isopropyl-β-D-thiogalactopyranoside) or such as an antibiotic(tetracycline, ampicillin, rifampicin, kanamycin), hormone (e.g.gibberellin, auxin, cytokinin, glucocorticoid, brassinosteroid,ethylene, abscisic acid etc), hormone analogue (indolacefic acid (IAA),2,4-D, etc), metal (zinc, copper, iron, etc), or dexamethasone, amongstothers. As will be known to those skilled in the art, expression of afunctional protein may also require one or more post-translationalmodifications, such as glycosylation, phosphorylation,dephosphorylation, or one or more protein-protein interactions, amongstothers. All such processes are included within the scope of the term“expressible format”.

Preferably, expression of a protein in a specific cell, tissue, ororgan, preferably of plant origin, is effected by introducing andexpressing an isolated nucleic acid molecule encoding said protein, suchas a cDNA molecule, genomic gene, synthetic oligonucleotide molecule,mRNA molecule or open reading frame, to said cell, tissue or organ,wherein said nucleic acid molecule is placed operably in connection withsuitable regulatory or control sequences including a promoter,preferably a plant-expressible promoter, and a terminator sequence.

Reference herein to a “promoter” is to be taken in its broadest contextand includes the transcriptional regulatory sequences derived from aclassical eukaryotic genomic gene, including the TATA box which isrequired for accurate transcription initiation, with or without a CCAATbox sequence and additional regulatory or control elements (i.e.upstream activating sequences, enhancers and silencers) which alter geneexpression in response to developmental and/or external stimuli, or in atissue-specific manner.

The term “promoter” also includes the transcriptional regulatorysequences of a classical prokaryotic gene, in which case it may includea −35 box sequence and/or a −10 box transcriptional regulatorysequences.

The term “promoter” is also used to describe a synthetic or fusionmolecule, or derivative which confers, activates or enhances expressionof a nucleic acid molecule in a cell, tissue or organ.

Promoters may contain additional copies of one or more specificregulatory elements, to further enhance expression and/or to alter thespatial expression and/or temporal expression of a nucleic acid moleculeto which it is operably connected. Such regulatory elements may beplaced adjacent to a heterologous promoter sequence to drive expressionof a nucleic acid molecule in response to e.g. copper, glucocorticoids,dexamethasone, tetracycline, gibberellin, cAMP, abscisic acid, auxin,wounding, ethylene, jasmonate or salicylic acid or to confer expressionof a nucleic acid molecule to specific cells, tissues or organs such asmeristems, leaves, roots, embryo, flowers, seeds or fruits.

In the context of the present invention, the promoter preferably is aplant-expressible promoter sequence. Promoters that also function orsolely function in non-plant cells such as bacteria, yeast cells, insectcells and animal cells are not excluded from the invention. By“plant-expressible” is meant that the promoter sequence, including anyadditional regulatory elements added thereto or contained therein, is atleast capable of inducing, conferring, activating or enhancingexpression in a plant cell, tissue or organ, preferably amonocotyledonous or dicotyledonous plant cell, tissue, or organ.

The terms “plant-operable” and “operable in a plant” when used herein,in respect of a promoter sequence, shall be taken to be equivalent to aplant-expressible promoter sequence.

Regulatable promoters as part of a binary viral plant expression systemare also known to the skilled artisan (Yadav 1999—WO9922003; Yadav2000—WO0017365).

In the present context, a “regulatable promoter sequence” is a promoterthat is capable of conferring expression on a structural gene in aparticular cell, tissue, or organ or group of cells, tissues or organsof a plant, optionally under specific conditions, however does generallynot confer expression throughout the plant under all conditions.Accordingly, a regulatable promoter sequence may be a promoter sequencethat confers expression on a gene to which it is operably connected in aparticular location within the plant or alternatively, throughout theplant under a specific set of conditions, such as following induction ofgene expression by a chemical compound or other elicitor.

Preferably, the regulatable promoter used in the performance of thepresent invention confers expression in a specific location within theplant, either constitutively or following induction, however not in thewhole plant under any circumstances. Included within the scope of suchpromoters are cell-specific promoter sequences, tissue-specific promotersequences, organ-specific promoter sequences, cell cycle specific genepromoter sequences, inducible promoter sequences and constitutivepromoter sequences that have been modified to confer expression in aparticular part of the plant at any one time, such as by integration ofsaid constitutive promoter within a transposable genetic element (Ac,Ds, Spm, En, or other transposon).

Similarly, the term “tissue-specific” shall be taken to indicate thatexpression is predominantly in a particular tissue or tissue-type,preferably of plant origin, albeit not necessarily exclusively in saidtissue or tissue-type.

Similarly, the term “organ-specific” shall be taken to indicate thatexpression is predominantly in a particular organ, preferably of plantorigin, albeit not necessarily exclusively in said organ.

Similarly, the term “cell cycle specific” shall be taken to indicatethat expression is predominantly cyclic and occurring in one or more,not necessarily consecutive phases of the cell cycle albeit notnecessarily exclusively in cycling cells, preferably of plant origin.

Those skilled in the art will be aware that an “inducible promoter” is apromoter the transcriptional activity of which is increased or inducedin response to a developmental, chemical, environmental, or physicalstimulus. Similarly, the skilled craftsman will understand that a“constitutive promoter” is a promoter that is transcriptionally activethroughout most, but not necessarily all parts of an organism,preferably a plant, during most, but not neccessarily all phases of itsgrowth and development.

Those skilled in the art will readily be capable of selectingappropriate promoter sequences for use in regulating appropriateexpression of the cytokinin oxidase protein from publicly-available orreadily-available sources, without undue experimentation. Placing anucleic acid molecule under the regulatory control of a promotersequence, or in operable connection with a promoter sequence, meanspositioning said nucleic acid molecule such that expression iscontrolled by the promoter sequence. A promoter is usually, but notnecessarily, positioned upstream, or at the 5′-end, and within 2 kb ofthe start site of transcription, of the nucleic acid molecule which itregulates. In the construction of heterologous promoter/structural genecombinations it is generally preferred to position the promoter at adistance from the gene transcription start site that is approximatelythe same as the distance between that promoter and the gene it controlsin its natural setting (i.e., the gene from which the promoter isderived). As is known in the art, some variation in this distance can beaccommodated without loss of promoter function. Similarly, the preferredpositioning of a regulatory sequence element with respect to aheterologous gene to be placed under its control is defined by thepositioning of the element in its natural setting (i.e., the gene fromwhich it is derived). Again, as is known in the art, some variation inthis distance can also occur.

Examples of promoters suitable for use in gene constructs of the presentinvention include those listed in Table 4, amongst others. The promoterslisted in Table 4 are provided for the purposes of exemplification onlyand the present invention is not to be limited by the list providedtherein. Those skilled in the art will readily be in a position toprovide additional promoters that are useful in performing the presentinvention.

In the case of constitutive promoters or promoters that induceexpression throughout the entire plant, it is preferred that suchsequences are modified by the addition of nucleotide sequences derivedfrom one or more of the tissue-specific promoters listed in Table 4, oralternatively, nucleotide sequences derived from one or more of theabove-mentioned tissue-specific inducible promoters, to confertissue-specificity thereon. For example, the CaMV 35S promoter may bemodified by the addition of maize Adh1 promoter sequence, to conferanaerobically-regulated root-specific expression thereon, as describedpreviously (Ellis et al., 1987). Another example describes conferringroot specific or root abundant gene expression by fusing the CaMV35Spromoter to elements of the maize glycine-rich protein GRP3 gene (Feixand Wulff 2000—WO0015662). Such modifications can be achieved by routineexperimentation by those skilled in the art.

The term “terminator” refers to a DNA sequence at the end of atranscriptional unit which signals termination of transcription.Terminators are 3′-non-translated DNA sequences containing apolyadenylation signal, which facilitates the addition of polyadenylatesequences to the 3′-end of a primary transcript. Terminators active incells derived from viruses, yeasts, moulds, bacteria, insects, birds,mammals and plants are known and described in the literature. They maybe isolated from bacteria, fungi, viruses, animals and/or plants.

TABLE 4 Exemplary plant-expressible promoters for use in the performanceof the present invention I: CELL-SPECIFIC, TISSUE-SPECIFIC, ANDORGAN-SPECIFIC PROMOTERS EXPRESSION GENE SOURCE PATTERN REFERENCEα-amylase (Amy32b) aleurone Lanahan, M.B., et al., Plant Cell 4:203–211, 1992; Skriver, K, et al. Proc. Natl. Acad. Sci. (USA) 88:7266–7270, 1991 cathepsin β-like gene aleurone Cejudo, F. J., et al.,Plant Molecular Biology 20: 849–856, 1992. Agrobacterium cambium Nilssonet al., Physiol. Plant. 100: 456–462, rhizogenes rolB 1997 AtPRP4flowers http://salus.medium.edu/mmg/tierney/html chalcone synthaseflowers Van der Meer, et al., Plant Mol. Biol. (chsA) 15, 95–109, 1990.LAT52 anther Twell et al Mol. Gen Genet. 217: 240–245 (1989) apetala-3flowers chitinase fruit (berries, Thomas et al. CSIRO Plant Industry,grapes, etc) Urrbrae, South Australia, Australia;http://winetitles.com.au/gwrdc/csh95-1.html rbcs-3A green tissue (egLam, E. et al., The Plant Cell 2: 857–866, leaf) 1990.; Tucker et al.,Plant Physiol. 113: 1303–1308, 1992. leaf-specific genes leafBaszczynski, et al., Nucl. Acid Res. 16: 4732, 1988. AtPRP4 leafhttp://salus.medium.edu/mmg/tierney/html chlorella virus adenine leafMitra and Higgins, 1994, Plant methyltransferase gene Molecular Biology26: 85–93 promoter aldP gene promoter leaf Kagaya et al., 1995,Molecular and from rice General Genetics 248: 668–674 rbcs promoter fromrice leaf Kyozuka et al., 1993, Plant or tomato Physiology 102: 991–1000Pinus cab-6 leaf Yamamoto et al., Plant Cell Physiol. 35: 773–778, 1994.rubisco promoter leaf cab (chlorophyll leaf a/b/binding protein SAM22senescent leaf Crowell, et al., Plant Mol. Biol. 18: 459–466, 1992. ltpgene (lipid transfer Fleming, et al, Plant J. 2, 855–862. gene) R.japonicum nif gene Nodule U.S. Pat. No. 4, 803, 165 B. japonicum nifHgene Nodule U.S. Pat. No. 5, 008, 194 GmENOD40 Nodule Yang, et al., ThePlant J. 3: 573–585. PEP carboxylase Nodule Pathirana, et al., PlantMol. Biol. 20: (PEPC) 437–450, 1992. leghaemoglobin (Lb) Nodule Gordon,et al., J. Exp. Bot. 44: 1453–1465, 1993. Tungro bacilliform virusphloem Bhattacharyya-Pakrasi, et al, The gene Plant J. 4: 71–79, 1992.pollen-specific genes pollen; microspore Albani, et al., Plant Mol.Biol. 15: 605, 1990; Albani, et al., Plant Mol. Biol. 16: 501, 1991)Zm13 pollen Guerrero et al Mol. Gen. Genet. 224: 161–168 (1993) apg genemicrospore Twell et al Sex. Plant Reprod. 6: 217–224 (1993) maizepollen-specific pollen Hamilton, et al., Plant Mol. Biol. 18: gene211–218, 1992. sunflower pollen- pollen Baltz, et al., The Plant J. 2:713–721, expressed gene 1992. B. napus pollen- pollen; anther; Arnoldo,et al., J. Cell. Biochem., specific gene tapetum Abstract No. Y101, 204,1992. root-expressible genes roots Tingey, et al., EMBO J. 6: 1, 1987.tobacco auxin-inducible root tip Van der Zaal, et al., Plant Mol. Biol.gene 16, 983, 1991. β-tubulin root Oppenheimer, et al., Gene 63: 87,1988. tobacco root-specific root Conkling, et al., Plant Physiol. 93:genes 1203, 1990. B. napus G1-3b gene root U.S. Pat. No. 5, 401, 836SbPRP1 roots Suzuki et al., Plant Mol. Biol. 21: 109–119 1993. AtPRP1;AtPRP3 roots; root hairs http://salus.medium.edu/mmg/tierney/html RD2gene root cortex http://cnsu.edu/ncsu/research TobRB7 gene rootvasculature http://cnsu.edu/ncsu/research AtPRP4 leaves; flowers;http://salus.medium.edu/mmg/tierney/html lateral root primordiaseed-specific genes seed Simon, et al., Plant Mol. Biol. 5: 191, 1985;Scofield, et al., J. Biol. Chem. 262: 12202, 1987.; Baszczynski, et al.,Plant Mol. Biol. 14: 633, 1990. Brazil Nut albumin seed Pearson, et al.,Plant Mol. Biol. 18: 235–245, 1992. legumin seed Ellis, et al., PlantMol. Biol. 10: 203–214, 1988. glutelin (rice) seed Takaiwa, et al., Mol.Gen. Genet. 208: 15–22, 1986; Takaiwa, et al., FEBS Letts. 221: 43–47,1987. zein seed Matzke et al Plant Mol Biol, 14(3): 323–32 1990 napAseed Stalberg, et al, Planta 199: 515–519, 1996. wheat LMW and HMWendosperm Mol Gen Genet 216: 81–90, 1989; glutenin-1 NAR 17: 461–2, 1989wheat SPA seed Albani et al, Plant Cell, 9: 171–184, 1997 wheat α, β,γ-gliadins endosperm EMBO 3: 1409–15, 1984 barley ltr1 promoterendosperm barley B1, C, D, endosperm Theor Appl Gen 98: 1253–62, 1999;hordein Plant J 4: 343–55, 1993; Mol Gen Genet 250: 750–60, 1996 barleyDOF endosperm Mena et al, The Plant Journal, 116(1): 53–62, 1998 blz2endosperm EP99106056.7 synthetic promoter endosperm Vicente-Carbajosa etal., Plant J. 13: 629–640, 1998. rice prolamin NRP33 endosperm Wu et al,Plant Cell Physiology 39(8) 885–889, 1998 rice α-globulin Glb-1endosperm Wu et al, Plant Cell Physiology 39(8) 885–889, 1998 rice OSH1embryo Sato et al, Proc. Natl. Acad. Sci. USA, 93: 8117–8122, 1996 riceα-globulin endosperm Nakase et al. Plant Mol. Biol. 33: 513–522REB/OHP-1 1997 rice ADP-glucose PP endosperm Trans Res 6: 157–68, 1997maize ESR gene family endosperm Plant J 12: 235–46, 1997 sorgumγ-kafirin endosperm PMB 32: 1029–35, 1996 KNOX embryo Postma-Haarsma etal, Plant Mol. Biol. 39: 257–71, 1999 rice oleosin embryo and aleuron Wuet at, J. Biochem., 123: 386, 1998 sunflower oleosin seed (embryo andCummins, et al., Plant Mol. Biol. 19: dry seed) 873–876, 1992 LEAFYshoot meristem Weigel et al., Cell 69: 843–859, 1992. Arabidopsisthaliana shoot meristem Accession number AJ131822 knat1 Malus domesticakn1 shoot meristem Accession number Z71981 CLAVATA1 shoot meristemAccession number AF049870 stigma-specific genes stigma Nasrallah, etal., Proc. Natl. Acad. Sci. USA 85: 5551, 1988; Trick, et al., PlantMol. Biol. 15: 203, 1990. class I patatin gene tuber Liu et al., PlantMol. Biol. 153: 386–395, 1991. PCNA rice meristem Kosugi et al, NucleicAcids Research 19: 1571–1576, 1991; Kosugi S. and Ohashi Y, Plant Cell9: 1607–1619, 1997. Pea TubA1 tubulin Dividing cells Stotz and Long,Plant Mol.Biol. 41, 601–614. 1999 Arabidopsis cdc2a cycling cells Chungand Parish, FEBS Lett, 3; 362(2): 215–9, 1995 Arabidopsis Rop1A Anthers;mature Li et al. 1998 Plant Physiol 118, 407–417. pollen + pollen tubesArabidopsis AtDMC1 Meiosis-associated Klimyuk and Jones 1997 Plant J.11, 1–14. Pea PS-IAA4/5 and Auxin-inducible Wong et al. 1996 Plant J. 9,587–599. PS-IAA6 Pea Meristematic Zhou et al. 1997 Plant J. 12, 921–930farnesyltransferase tissues; phloem near growing tissues; light-andsugar-repressed Tobacco (N. sylvestris) Dividing cells/ Trehin et al.1997 Plant Mol.Biol. 35, cyclin B1; 1 meristematic tissue 667–672.Catharanthus roseus Dividing cells/ Ito et al. 1997 Plant J. 11, 983–992Mitotic cyclins CYS (A- meristematic tissue type) and CYM (B-type)Arabidopsis cyc1 At Dividing cells/ Shaul et al. 1996 (=cyc B1; 1) andmeristematic tissue Proc.Natl.Acad.Sci.U.S.A 93, 4868–4872. cyc3aAt(A-type) Arabidopsis tef1 Dividing cells/ Regad et al. 1995Mol.Gen.Genet. promoter box meristematic tissue 248, 703–711.Catharanthus roseus Dividing cells/ Ito et al. 1994 Plant Mol.Biol. 24,863–878. cyc07 meristematic tissue II: EXEMPLARY CONSTITUTIVE PROMOTERSEXPRESSION GENE SOURCE PATTERN REFERENCE Actin constitutive McElroy etal, Plant Cell, 2: 163–171, 1990 CAMV 35S constitutive Odell et al,Nature, 313: 810–812, 1985 CaMV 19S constitutive Nilsson et al.,Physiol. Plant. 100: 456–462, 1997 GOS2 constitutive de Pater et al,Plant J. 2: 837–844, 1992 ubiquitin constitutive Christensen et al,Plant Mol. Biol. 18: 675–689, 1992 rice cyclophilin constitutiveBuchholz et al, Plant Mol Biol. 25: 837–843, 1994 maize histone H3constitutive Lepetit et al, Mol. Gen. Genet. 231: 276–285, 1992 alfalfahistone H3 constitutive Wu et al., Nucleic Acids Res. 17: 3057–3063,1989; Wu et al., Plant Mol. Biol. 11: 641–649, 1988 actin 2 constitutiveAn et al, Plant J. 10(1); 107–121, 1996 III: EXEMPLARY STRESS-INDUCIBLEPROMOTERS NAME STRESS REFERENCE P5CS (delta(1)- salt, water Zhang et al.Plant Science. 129: 81–89, pyrroline-5-carboxylate 1997 syntase) cor15acold Hajela et al., Plant Physiol. 93: 1246–1252, 1990 cor15b coldWlihelm et al., Plant Mol Biol. 23: 1073–1077, 1993 cor15a (−305 to + 78nt) cold, drought Baker et al., Plant Mol Biol. 24: 701–713, 1994 rd29salt, drought, cold Kasuga et al., Nature Biotechnology 18: 287–291,1999 heat shock proteins, heat Barros et al., Plant Mol Biol 19: 665–75,including artificial 1992. Marrs et al., Dev promoters containing Genet14: 27–41, 1993. Schoffl et al., the heat shock element Mol Gen Gent,217: 246–53, 1989. (HSE) smHSP (small heat heat Waters et al, JExperimental Botany shock proteins) 47: 325–338, 1996 wcs120 coldOuellet et al., FEBS Lett. 423: 324–328, 1998 ci7 cold Kirch et al.,Plant Mol Biol 33: 897–909, 1997 Adh cold, drought, hypoxia Dolferus etal., Plant Physiol 105: 1075–87, 1994 pwsi18 water: salt and droughtJoshee et al., Plant Cell Physiol 39: 64–72, 1998 ci21A cold Schneideret al., Plant Physiol 113: 335–45, 1997 Trg-31 drought Chaudhary et al.,Plant Mol Biol 30: 1247–57, 1996 osmotin osmotic Raghothama et al.,Plant Mol Biol 23: 1117–28, 1993 Rab17 osmotic, ABA Vilardell et al.,Plant Mol Biol 17: 985–93, 1991 lapA wounding, enviromental WO99/03977University of California/INRA IV: EXEMPLARY PATHOGEN-INDUCIBLE PROMOTERSNAME PATHOGEN REFERENCE RB7 Root-knot nematodes US5760386 - NorthCarolina State (Meloidogyne spp.) University; Opperman et al (1994)Science 263: 221–23. PR-1, 2, 3, 4, 5, 8, 11 fungal, viral, bacterialWard et al (1991) Plant Cell 3: 1085–1094; Reiss et al 1996; Lebel et al(1998), Plant J, 16(2): 223–33; Melchers et al (1994), Plant J, 5(4):469–80; Lawton et al (1992), Plant Mol Biol, 19(5): 735–43. HMG2nematodes WO9503690 - Virginia Tech Intellectual Properties Inc. Abi3Cyst nematodes Unpublished (Heterodera spp.) ARM1 nematodes Barthels etal., (1997) The Plant Cell 9, 2119–2134. WO 98/31822 —Plant GeneticSystems Att0728 nematodes Barthels et al., (1997) The Plant Cell 9,2119–2134. PCT/EP98/07761 Att1712 nematodes Barthels et al., (1997) ThePlant Cell 9, 2119–2134. PCT/EP98/07761 Gst1 Different types ofStrittmatter et al (1996) Mol. pathogens Plant-Microbe Interact. 9,68–73. LEMMI nematodes WO 92/21757 - Plant Genetic Systems CLEgeminivirus PCT/EP99/03445 - CINESTAV PDF1.2 Fungal Including Manners etal (1998), Plant Mol Altemaria brassicicola Biol, 38(6): 1071–80. andBotrytis cinerea Thi2.1 Fungal - Fusarium Vignutelli et al (1998) Plantoxysporum f sp. J; 14(3): 285–95 matthiolae DB#226 nematodes Bird andWilson (1994) Mol. Plant- Microbe Interact., 7, 419–42 WO 95.322888DB#280 nematodes Bird and Wilson (1994) Mol. Plant- Microbe Interact.,7, 419–42 WO 95.322888 Cat2 nematodes Niebel et at (1995) Mol PlantMicrobe Interact 1995 May–Jun; 8(3): 371–8 □Tub nematodes Aristizabal etal (1996), 8^(th) International Congress on Plant- Microbe Interaction,Knoxville US B-29 SHSP nematodes Fenoll et al (1997) In: Cellular andmolecular aspects of plant- nematode interactions. Kluwer Academic, C.Fenoll, F.M.W. Grundler and S.A. Ohl (Eds.), Tsw12 nematodes Fenoll etal (1997) In: Cellular and molecular aspects of plant- nematodeinteractions. Kluwer Academic, C. Fenoll, F.M.W. Grundler and S. A. Ohl(Eds.) Hs1(pro1) nematodes WO 98/122335 - Jung NsLTP viral, fungal,bacterial Molina & Garc ia-Olmedo (1993) FEBS Lett, 316(2): 119–22 RIPviral, fungal Tumer et al (1997) Proc Natl Acad Sci U.S.A, 94(8):3866–71

Examples of terminators particularly suitable for use in the geneconstructs of the present invention include the Agrobacteriumtumefaciens nopaline synthase (NOS) gene terminator, the Agrobacteriumtumefaciens octopine synthase (OCS) gene terminator sequence, theCauliflower mosaic virus (CaMV) 35S gene terminator sequence, the Oryzasativa ADP-glucose pyrophosphorylase terminator sequence (t3′Bt2), theZea mays zein gene terminator sequence, the rbcs-1A gene terminator, andthe rbcs-3A gene terminator sequences, amongst others.

Preferred promoter sequences of the invention include root specificpromoters such as but not limited to the ones listed in Table 5 and asoutlined in the Examples.

TABLE 5 Exemplary root specific promoters for use in the performance ofthe present invention NAME ORIGIN REFERENCE SbPRP1 Soybean Suzuki etal., Plant Mol Biol, 21: 109–119, 1993 636 bp fragment of TobaccoYamamoto et al., Plant Cell TobRB7 3: 371–382, 1991 GGPS3 ArabidopsisOkada et al., Plant Physiol 122: 1045–1056, 2000 580 bp fragmentArabidopsis Wanapu and Shinmyo, Ann N Y of prxEa Acad Sci 782: 107–114,1996 Ids2 promoter Barley Okumura et al., Plant Mol Biol 25: 705–719,1994 AtPRP3 Arabidopsis Fowler et al., Plant Physiol 121: 1081–1092,1999

Those skilled in the art will be aware of additional promoter sequencesand terminator sequences which may be suitable for use in performing theinvention. Such sequences may readily be used without any undueexperimentation.

In the context of the current invention, “ectopic expression” or“ectopic overexpression” of a gene or a protein are conferring toexpression patterns and/or expression levels of said gene or proteinnormally not occurring under natural conditions, more specifically ismeant increased expression and/or increased expression levels. Ectopicexpression can be achieved in a number of ways including operablylinking of a coding sequence encoding said protein to an isolatedhomologous or heterologous promoter in order to create a chimeric geneand/or operably linking said coding sequence to its own isolatedpromoter (i.e. the unisolated promoter naturally driving expression atsaid protein) in order to create a recombinant gene duplication or genemultiplication effect. With “ectopic co-expression” is meant the ectopicexpression or ectopic overexpression of two or more genes or proteins.The same or, more preferably, different promoters are used to conferectopic expression of said genes or proteins.

Preferably, the promoter sequence used in the context of the presentinvention is operably linked to a coding sequence or open reading frame(ORF) encoding a cytokinin oxidase protein or a homologue, derivative oran immunologically active and/or functional fragment thereof as definedsupra.

“Downregulation of expression” as used herein means lowering levels ofgene expression and/or levels of active gene product and/or levels ofgene product activity. Decreases in expression may be accomplished bye.g. the addition of coding sequences or parts thereof in a senseorientation (if resulting in co-suppression) or in an antisenseorientation relative to a promoter sequence and furthermore by e.g.insertion mutagenesis (e.g. T-DNA insertion or transposon insertion) orby gene silencing strategies as described by e.g. Angell and Baulcombe(1998—WO9836083), Lowe et al. (1989—WO9853083), Lederer et al.(1999—WO9915682) or Wang et al. (1999—WO9953050). Genetic constructsaimed at silencing gene expression may have the nucleotide sequence ofsaid gene (or one or more parts thereof) contained therein in a senseand/or antisense orientation relative to the promoter sequence. Anothermethod to downregulate gene expression comprises the use of ribozymes.

Modulating, including lowering, the level of active gene products or ofgene product activity can be achieved by administering or exposingcells, tissues, organs or organisms to said gene product, a homologue,derivative and/or immunologically active fragment thereof.Immunomodulation is another example of a technique capable ofdownregulation levels of active gene product and/or of gene productactivity and comprises administration of or exposing to or expressingantibodies to said gene product to or in cells, tissues, organs ororganisms wherein levels of said gene product and/or gene productactivity are to be modulated. Such antibodies comprise “plantibodies”,single chain antibodies, IgG antibodies and heavy chain camel antibodiesas well as fragments thereof.

Modulating, including lowering, the level of active gene products or ofgene product activity can futhermore be achieved by administering orexposing cells, tissues, organs or organisms to an agonist of said geneproduct or the activity thereof. Such agonists include proteins(comprising e.g. kinases and proteinases) and chemical compoundsidentified according to the current invention as described supra.

In the context of the current invention is envisaged the downregulationof the expression of a cytokinin oxidase gene as defined higher.Preferably said cytokinin oxidase gene is a plant cytokinin oxidasegene, more specifically an AtCKX. The invention further comprisesdownregulation of levels of a cytokinin oxidase protein or of acytokinin oxidase activity whereby said cytokinin oxidase protein hasbeen defined supra. Preferably said cytokinin oxidase protein is a plantcytokinin oxidase, more specifically an AtCKX.

By “modifying cell fate and/or plant development and/or plant morphologyand/or biochemistry and/or physiology” is meant that one or moredevelopmental and/or morphological and/or biochemical and/orphysiological characteristics of a plant is altered by the performanceof one or more steps pertaining to the invention described herein.

“Cell fate” refers to the cell-type or cellular characteristics of aparticular cell that are produced during plant development or a cellularprocess therefor, in particular during the cell cycle or as aconsequence of a cell cycle process.

“Plant development” or the term “plant developmental characteristic” orsimilar term shall, when used herein, be taken to mean any cellularprocess of a plant that is involved in determining the developmentalfate of a plant cell, in particular the specific tissue or organ typeinto which a progenitor cell will develop. Cellular processes relevantto plant development will be known to those skilled in the art. Suchprocesses include, for example, morphogenesis, photomorphogenesis, shootdevelopment, root development, vegetative development, reproductivedevelopment, stem elongation, flowering, and regulatory mechanismsinvolved in determining cell fate, in particular a process or regulatoryprocess involving the cell cycle.

“Plant morphology” or the term “plant morphological characteristic” orsimilar term will, when used herein, be understood by those skilled inthe art to refer to the external appearance of a plant, including anyone or more structural features or combination of structural featuresthereof. Such structural features include the shape, size, number,position, colour, texture, arrangement, and patternation of any cell,tissue or organ or groups of cells, tissues or organs of a plant,including the root, stem, leaf, shoot, petiole, trichome, flower, petal,stigma, style, stamen, pollen, ovule, seed, embryo, endosperm, seedcoat, aleurone, fibre, fruit, cambium, wood, heartwood, parenchyma,aerenchyma, sieve element, phloem or vascular tissue, amongst others.

“Plant biochemistry” or the term “plant biochemical characteristic” orsimilar term will, when used herein, be understood by those skilled inthe art to refer to the metabolic and catalytic processes of a plant,including primary and secondary metabolism and the products thereof,including any small molecules, macromolecules or chemical compounds,such as but not limited to starches, sugars, proteins, peptides,enzymes, hormones, growth factors, nucleic acid molecules, celluloses,hemicelluloses, calloses, lectins, fibres, pigments such asanthocyanins, vitamins, minerals, micronutrients, or macronutrients,that are produced by plants.

“Plant physiology” or the term “plant physiological characteristic” orsimilar term will, when used herein, be understood to refer to thefunctional processes of a plant, including developmental processes suchas growth, expansion and differentiation, sexual development, sexualreproduction, seed set, seed development, grain filling, asexualreproduction, cell division, dormancy, germination, light adaptation,photosynthesis, leaf expansion, fibre production, secondary growth orwood production, amongst others; responses of a plant toexternally-applied factors such as metals, chemicals, hormones, growthfactors, environment and environmental stress factors (eg. anoxia,hypoxia, high temperature, low temperature, dehydration, light,daylength, flooding, salt, heavy metals, amongst others), includingadaptive responses of plants to said externally-applied factors.

Means for introducing recombinant DNA into plant tissue or cellsinclude, but are not limited to, transformation using CaCl₂ andvariations thereof, in particular the method described by Hanahan(1983), direct DNA uptake into protoplasts (Krens et al, 1982;Paszkowski et al, 1984), PEG-mediated uptake to protoplasts (Armstronget al, 1990) microparticle bombardment, electroporation (Fromm et al.,1985), microinjection of DNA (Crossway et al, 1986), microparticlebombardment of tissue explants or cells (Christou et al, 1988; Sanford,1988), vacuum-infiltration of tissue with nucleic acid, or in the caseof plants, T-DNA-mediated transfer from Agrobacterium to the planttissue as described essentially by An et al. (1985), Dodds et al.,(1985), Herrera-Estrella et at (1983a, 1983b, 1985). Methods fortransformation of monocotyledonous plants are well known in the art andinclude Agrobacterium-mediated transformation (Cheng et al.,1997—WO9748814; Hansen 1998—WO9854961; Hiel et at, 1994—WO9400977; Hielet at, 1998—WO9817813; Rikllshi et al., 1999—WO9904618; Saito et al,1995—WO9506722), microprojectile bombardment (Adams et al., 1999—U.S.Pat. No. 5,969,213; Bowen et al, 1998—U.S. Pat. No. 5,736,369; Chang etal., 1994—WO9413822; Lundquist et al., 1999—U.S. Pat. No. 5,874,265/U.S.Pat. No. 5,990,390; Vasil and Vasil, 1995—U.S. Pat. No. 5,405,765.Walker et al., 1999—U.S. Pat. No. 5,955,362), DNA uptake (Eyal et al.,1993—WO9318168), microinjection of Agrobacterium cells (von Holt,1994—DE4309203) and sonication (Finer et al., 1997—U.S. Pat. No.5,693,512).

For microparticle bombardment of cells, a microparticle is propelledinto a cell to produce a transformed cell. Any suitable ballistic celltransformation methodology and apparatus can be used in performing thepresent invention. Exemplary apparatus and procedures are disclosed byStomp et al. (U.S. Pat. No. 5,122,466) and Sanford and Wolf (U.S. Pat.No. 4,945,050). When using ballistic transformation procedures, the geneconstruct may incorporate a plasmid capable of replicating in the cellto be transformed. Examples of microparticles suitable for use in suchsystems include 1 to 5 μm gold spheres. The DNA construct may bedeposited on the microparticle by any suitable technique, such as byprecipitation.

A whole plant may be regenerated from the transformed or transfectedcell, in accordance with procedures well known in the art. Plant tissuecapable of subsequent clonal propagation, whether by organogenesis orembryogenesis, may be transformed with a gene construct of the presentinvention and a whole plant regenerated therefrom. The particular tissuechosen will vary depending on the clonal propagation systems availablefor, and best suited to, the particular species being transformed.Exemplary tissue targets include leaf disks, pollen, embryos,cotyledons, hypocotyls, megagametophytes, callus tissue, existingmeristematic tissue (e.g., apical meristem, axillary buds, and rootmeristems), and induced meristem tissue (e.g., cotyledon meristem andhypocotyl meristem).

The term “organogenesis”, as used herein, means a process by whichshoots and roots are developed sequentially from meristematic centres.

The term “embryogenesis”, as used herein, means a process by whichshoots and roots develop together in a concerted fashion (notsequentially), whether from somatic cells or gametes.

Preferably, the plant is produced according to the inventive method istransfected or transformed with a genetic sequence, or amenable to theintroduction of a protein, by any art-recognized means, such asmicroprojectile bombardment, microinjection, Agrobacterium-mediatedtransformation (including in planta transformation), protoplast fusion,or electroporation, amongst others. Most preferably said plant isproduced by Agrobacterium-mediated transformation.

Agrobacterium-mediated transformation or agrolistic transformation ofplants, yeast, moulds or filamentous fungi is based on the transfer ofpart of the transformation vector sequences, called the T-DNA, to thenucleus and on integration of said T-DNA in the genome of saideukaryote.

With “Agrobacterium” is meant a member of the Agrobacteriaceae, morepreferably Agrobacterium or Rhizobactedum and most preferablyAgrobacterium tumefaciens.

With “T-DNA”, or transferred DNA, is meant that part of thetransformation vector flanked by T-DNA borders which is, afteractivation of the Agrobacterium vir genes, nicked at the T-DNA bordersand is transferred as a single stranded DNA to the nucleus of aneukaryotic cell.

When used herein, with “T-DNA borders”, “T-DNA border region”, or“border region” are meant either right T-DNA border (RB) or left T-DNAborder (LB). Such a border comprises a core sequence flanked by a borderinner region as part of the T-DNA flanking the border and/or a borderouter region as part of the vector backbone flanking the border. Thecore sequences comprise 22 bp in case of octopine-type vectors and 25 bpin case of nopaline-type vectors. The core sequences in the right borderregion and left border region form imperfect repeats. Border coresequences are indispensable for recognition and processing by theAgrobacterium nicking complex consisting of at least VirD1 and VirD2.Core sequences flanking a T-DNA are sufficient to promote transfer ofsaid T-DNA. However, efficiency of transformation using transformationvectors carrying said T-DNA solely flanked by said core sequences islow. Border inner and outer regions are known to modulate efficiency ofT-DNA transfer (Wang et al. 1987). One element enhancing T-DNA transferhas been characterized and resides in the right border outer region andis called overdrive (Peralta et al. 1986, van Haaren et al. 1987).

With “T-DNA transformation vector” or “T-DNA vector” is meant any vectorencompassing a T-DNA sequence flanked by a right and left T-DNA borderconsisting of at least the right and left border core sequences,respectively, and used for transformation of any eukaryotic cell.

With “T-DNA vector backbone sequence” or “T-DNA vector backbonesequences” is meant all DNA of a T-DNA containing vector that liesoutside of the T-DNA borders and, more specifically, outside the nickingsites of the border core imperfect repeats.

The current invention includes optimized T-DNA vectors such that vectorbackbone integration in the genome of a eukaryotic cell is minimized orabsent. With “optimized T-DNA DNA vector” is meant a T-DNA vectordesigned either to decrease or abolish transfer of vector backbonesequences to the genome of a eukaryotic cell. Such T-DNA vectors areknown to the one familiar with the art and include those described byHanson et al. (1999) and by Stuiver et al. (1999—WO9901563).

The current invention clearly considers the inclusion of a DNA sequenceencoding a cytokinin oxidase, homologue, derivative or immunologicallyactive and/or functional fragment thereof as defined supra, in any T-DNAvector comprising binary transformation vectors, super-binarytransformation vectors, co-integrate transformation vectors, Ri-derivedtransformation vectors as well as in T-DNA carrying vectors used inagrolistic transformation. Preferably, said cytokinin oxidase is a plantcytokinin oxidase, more specifically an Arabidopsis thaliana (At)CKX.

With “binary transformation vector” is meant a T-DNA transformationvector comprising:

-   -   (a) a T-DNA region comprising at least one gene of interest        and/or at least one selectable marker active in the eukaryotic        cell to be transformed; and    -   (b) a vector backbone region comprising at least origins of        replication active in E. coli and Agrobacterium and markers for        selection in E. coli and Agrobacterium.

The T-DNA borders of a binary transformation vector can be derived fromoctopine-type or nopaline-type Ti plasmids or from both. The T-DNA of abinary vector is only transferred to a eukaryotic cell in conjunctionwith a helper plasmid.

With “helper plasmid” is meant a plasmid that is stably maintained inAgrobacterium and is at least carrying the set of vir genes necessaryfor enabling transfer of the T-DNA. Said set of vir genes can be derivedfrom either octopine-type or nopaline-type Ti plasmids or from both.

With “super-binary transformation vector” is meant a binarytransformation vector additionally carrying in the vector backboneregion a vir region of the Ti plasmid pTiBo542 of the super-virulent A.tumefaciens strain A281 (EP0604662, EP0687730). Super-binarytransformation vectors are used in conjunction with a helper plasmid.

With “co-integrate transformation vector” is meant a T-DNA vector atleast comprising:

-   -   (a) a T-DNA region comprising at least one gene of interest        and/or at least one selectable marker active in plants; and    -   (b) a vector backbone region comprising at least origins of        replication active in Escherichia coli and Agrobacterium, and        markers for selection in E. coli and Agrobacterium, and a set of        vir genes necessary for enabling transfer of the T-DNA.

The T-DNA borders and said set of vir genes of a said T-DNA vector canbe derived from either octopine-type or nopaline-type Ti plasmids orfrom both.

With “Ri-derived plant transformation vector” is meant a binarytransformation vector in which the T-DNA borders are derived from a Tiplasmid and said binary transformation vector being used in conjunctionwith a ‘helper’ Ri-plasmid carrying the necessary set of vir genes.

As used herein, the term “selectable marker gene” or “selectable marker”or “marker for selection” includes any gene which confers a phenotype ona cell in which it is expressed to facilitate the identification and/orselection of cells which are transfected or transformed with a geneconstruct of the invention or a derivative thereof. Suitable selectablemarker genes contemplated herein include the ampicillin resistance(Amp′), tetracycline resistance gene (Tc′), bacterial kanamycinresistance gene (Kan′), phosphinothricin resistance gene, neomycinphosphotransferase gene (nptll), hygromycin resistance gene,β-glucuronidase (GUS) gene, chloramphenicol acetyltransferase (CAT)gene, green fluorescent protein (gfp) gene (Haseloff et al, 1997), andluciferase gene, amongst others.

With “agrolistics”, “agrolistic transformation” or “agrolistic transfer”is meant here a transformation method combining features ofAgrobacterium-mediated transformation and of biolistic DNA delivery. Assuch, a T-DNA containing target plasmid is co-delivered with DNA/RNAenabling in planta production of VirD1 and VirD2 with or without VirE2(Hansen and Chilton 1996; Hansen et al. 1997; Hansen and Chilton1997—WO9712046). With “foreign DNA” is meant any DNA sequence that isintroduced in the host's genome by recombinant techniques. Said foreignDNA includes e.g. a T-DNA sequence or a part thereof such as the T-DNAsequence comprising the selectable marker in an expressible format.Foreign DNA furthermore include intervening DNA sequences as definedsupra. With “recombination event” is meant either a site-specificrecombination event or a recombination event effected by transposon‘jumping’.

With “recombinase” is meant either a site-specific recombinase or atransposase.

With “recombination site” is meant either site-specific recombinationsites or transposon border sequences.

With “site specific recombination event” is meant an event catalyzed bya system generally consisting of three elements: a pair of DNA sequences(the site-specific recombination sequences or sites) and a specificenzyme (the site-specific recombinase). The site-specific recombinasecatalyzes a recombination reaction only between two site-specificrecombination sequences depending on the orientation of thesite-specific recombination sequences. Sequences intervening between twosite-specific recombination sites will be inverted in the presence ofthe site-specific recombinase when the site-specific recombinationsequences are oriented in opposite directions relative to one another(i.e. inverted repeats). If the site-specific recombination sequencesare oriented in the same direction relative to one another (i.e. directrepeats), then any intervening sequences will be deleted uponinteraction with the site-specific recombinase. Thus, if thesite-specific recombination sequences are present as direct repeats atboth ends of a foreign DNA sequence integrated into a eukaryotic genome,such integration of said sequences can subsequently be reversed byinteraction of the site-specific recombination sequences with thecorresponding site specific recombinase. A number of different sitespecific recombinase systems can be used including but not limited tothe Cre/lox system of bacteriophage P1, the FLP/FRT system of yeast, theGin recombinase of phage Mu, the Pin recombinase of E. coli, the PinB,PinD and PinF from Shigella, and the R/RS system of the pSR1 plasmid.Recombinases generally are integrases, resolvases or flippases. Alsodual-specific recombinases can be used in conjunction with direct orindirect repeats of two different site-specific recombination sitescorresponding to the dual-specific recombinase (WO99/25840). The twopreferred site-specific recombinase systems are the bacteriophage P1Cre/lox and the yeast FLP/FRT systems. In these systems a recombinase(Cre or FLP) interact specifically with its respective site-specificrecombination sequence (lox or FRT respectively) to invert or excise theintervening sequences. The site-specific recombination sequences foreach of these two systems are relatively short (34 bp for lox and 47 bpfor FRT). Some of these systems have already been used with highefficiency in plants such as tobacco (Dale et al. 1990) and Arabidopsis(Osborne et al. 1995). Site-specific recombination systems have manyapplications in plant molecular biology including methods for control ofhomologous recombination (e.g. U.S. Pat. No. 5,527,695), for targetedinsertion, gene stacking, etc. (WO99/25821) and for resolution ofcomplex T-DNA integration patterns or for excision of a selectablemarker (WO99/23202).

Although the site-specific recombination sequences must be linked to theends of the DNA to be excised or to be inverted, the gene encoding thesite specific recombinase may be located elsewhere. For example, therecombinase gene could already be present in the eukaryote's DNA orcould be supplied by a later introduced DNA fragment either introduceddirectly into cells, through crossing or through cross-pollination.Alternatively, a substantially purified recombinase protein could beintroduced directly into the eukaryotic cell, e.g. by micro-injection orparticle bombardment. Typically, the site-specific recombinase codingregion will be operably linked to regulatory sequences enablingexpression of the site-specific recombinase in the eukaryotic cell.

With “recombination event effected by transposon jumping” or“transposase-mediated recombination” is meant a recombination eventcatalyzed by a system consisting of three elements: a pair of DNAsequences (the transposon border sequences) and a specific enzyme (thetransposase). The transposase catalyzes a recombination reaction onlybetween two transposon border sequences which are arranged as invertedrepeats. A number of different transposon/transposase systems can beused including but not limited to the Ds/Ac system, the Spm system andthe Mu system. These systems originate from corn but it has been shownthat at least the Ds/Ac and the Spm system also function in other plants(Fedoroff et al. 1993, Schlappi et al. 1993, Van Sluys et al. 1987).Preferred are the Ds- and the Spm-type transposons which are delineatedby 11 bp- and 13 bp-border sequences, respectively.

Although the transposon border sequences must be linked to the ends ofthe DNA to be excised, the gene encoding the transposase may be locatedelsewhere. For example, the recombinase gene could already be present inthe eukaryote's DNA or could be supplied by a later introduced DNAfragment either introduced directly into cells, through crossing orthrough cross-pollination. Alternatively, a substantially purifiedtransposase protein could be introduced directly into cells, e.g. bymicroinjection or by particle bombardment.

As part of the current invention, transposon border sequences areincluded in a foreign DNA sequence such that they lie outside said DNAsequence and transform said DNA into a transposon-like entity that canmove by the action of a transposase.

As transposons often reintegrate at another locus of the host's genome,segregation of the progeny of the hosts in which the transposase wasallowed to act might be necessary to separate transformed hostscontaining e.g. only the transposon footprint and transformed hostsstill containing the foreign DNA.

In performing the present invention, the genetic element is preferablyinduced to mobilize, such as, for example, by the expression of arecombinase protein in the cell which contacts the integration site ofthe genetic element and facilitates a recombination event therein,excising the genetic element completely, or alternatively, leaving a“footprint”, generally of about 20 nucleotides in length or greater, atthe original integration site. Those hosts and host parts that have beenproduced according to the inventive method can be identified by standardnucleic acid hybridization and/or amplification techniques to detect thepresence of the mobilizable genetic element or a gene constructcomprising the same. Alternatively, in the case of transformed hostcells, tissues, and hosts wherein the mobilizable genetic element hasbeen excised, it is possible to detect a footprint in the genome of thehost which has been left following the excision event, using suchtechniques. As used herein, the term “footprint” shall be taken to referto any derivative of a mobilizable genetic element or gene constructcomprising the same as described herein which is produced by excision,deletion or other removal of the mobilizable genetic element from thegenome of a cell transformed previously with said gene construct. Afootprint generally comprises at least a single copy of therecombination loci or transposon used to promote excision. However, afootprint may comprise additional sequences derived from the geneconstruct, for example nucleotide sequences derived from the left bordersequence, right border sequence, origin of replication,recombinase-encoding or transposase-encoding sequence if used, or othervector-derived nucleotide sequences. Accordingly, a footprint isidentifiable according to the nucleotide sequence of the recombinationlocus or transposon of the gene construct used, such as, for example, asequence of nucleotides corresponding or complementary to a lox site orfit site.

The term “cell cycle” means the cyclic biochemical and structural eventsassociated with growth and with division of cells, and in particularwith the regulation of the replication of DNA and mitosis. Cell cycleincludes phases called: G0, Gap1 (G1), DNA synthesis (S), Gap2 (G2), andmitosis (M). Normally these four phases occur sequentially, however, thecell cycle also includes modified cycles wherein one or more phases areabsent resulting in modified cell cycle such as endomitosis,acytokinesis, polyploidy, polyteny, and endoreduplication.

The term “cell cycle progression” refers to the process of passingthrough the different cell cycle phases. The term “cell cycleprogression rate” accordingly refers to the speed at which said cellcycle phases are run through or the time spans required to complete saidcell cycle phases.

With “two-hybrid assay” is meant an assay that is based on theobservation that many eukaryotic transcription factors comprise twodomains, a DNA-binding domain (DB) and an activation domain (AD) which,when physically separated (i.e. disruption of the covalent linkage) donot effectuate target gene expression. Two proteins able to interactphysically with one of said proteins fused to DB and the other of saidproteins fused to AD will re-unite the DB and AD domains of thetranscription factor resulting in target gene expression. The targetgene in the yeast two-hybrid assay is usually a reporter gene such asthe β-galactosidase gene. Interaction between protein partners in theyeast two-hybrid assay can thus be quantified by measuring the activityof the reporter gene product (Bartel and Fields 1997). Alternatively, amammalian two-hybrid system can be used which includes e.g. a chimericgreen fluorescent protein encoding reporter gene (Shioda et al, 2000).

Furthermore, folding simulations and computer redesign of structuralmotifs of the protein of the invention can be performed usingappropriate computer programs (Olszewski, Proteins 25 (1996), 286–299;Hoffman, Comput. Appl. Biosci. 1 (1995), 675–679). Computer modeling ofprotein folding can be used for the conformational and energeticanalysis of detailed peptide and protein models (Monge, J. Mol. Biol.247 (1995), 995–1012; Renouf, Adv. Exp. Med. Biol. 376 (1995), 37–45).In particular, the appropriate programs can be used for theidentification of interactive sites of the cytokinin oxidases, itsligands or other interacting proteins by computer assistant searches forcomplementary peptide sequences (Fassina, Immunomethods 5 (1994),114–120). Further appropriate computer systems for the design of proteinand peptides are described in the prior art, for example in Berry,Biochem. Soc. Trans. 22 (1994), 1033–1036; Wodak, Ann, N.Y. Acac. Sci.501 (1987), 1–13; Pabo, Biochemistry 25 (1986), 5987–5991. The resultsobtained form the above-described computer analysis can be used for,e.g. the preparation of peptidomimetics of the protein of the inventionor fragments thereof. Such pseudopeptide analogues of the natural aminoacid sequence of the protein may very efficiently mimic the parentprotein (Benkirane, J. Biol. Chem, 271 (1996), 33218–33224). Forexample, incorporation of easily available achiral Ω-amino acid residuesinto a protein of the invention or a fragment thereof results in thesubstitution of amino bonds by polymethylene units of an aliphaticchain, thereby providing a convenient strategy for constructing apeptidomimetic (Banerjee, Biopolymers 39 (1996), 769–777). Superactivepeptidomimetic analogues of small peptide hormones in other systems aredescribed in the prior art (Zhang, Biochem. Biophys. Res. Commun. 224(1996), 327–331). Appropriate peptidomimetics of the protein of thepresent invention can also be identified by the synthesis ofpeptidomimetic combinatorial libraries through successive aminealkylation and testing the resulting compounds, e.g., for their binding,kinase inhibitory and/or immunlogical properties. Methods for thegeneration and use of peptidomimetic combinatorial libraries aredescribed in the prior art, for example in Ostresh, Methods inEnzymology 267 (1996), 220–234 and Dorner, Bioorg. Med. Chem. 4 (1996),709–715.

Furthermore, a three-dimensional and/or crystallographic structure ofthe protein of the invention can be used for the design ofpeptidomimetic inhibitors of the biological activity of the protein ofthe invention (Rose, Biochemistry 35 (1996), 12933–12944; Ruterber,Bioorg. Med. Chem. 4 (1996), 1545–1558).

The compounds to be obtained or identified in the methods of theinvention can be compounds that are able to bind to any of the nucleicacids, peptides or proteins of the invention. Other interestingcompounds to be identified are compounds that modulate the expression ofthe genes or the proteins of the invention in such a way that either theexpression of said gene or protein is enhanced or decreased by theaction of said compound. Alternatively the compound can exert his actionby enhancing or decreasing the activity of any of the proteins of theinvention. Herein, preferred proteins are novel cytokinin oxidases.

Said compound or plurality of compounds may be comprised in, forexample, samples, e.g., cell extracts from, e.g., plants, animals ormicroorganisms. Furthermore, said compound(s) may be known in the artbut hitherto not known to be capable of suppressing or activatingcytokinin oxidase interacting proteins. The reaction mixture may be acell free extract of may comprise a cell or tissue culture. Suitable setups for the method of the invention are known to the person skilled inthe art and are, for example, generally described in Alberts et al.,Molecular Biology of the Cell, third edition (1994), in particularChapter 17. The plurality of compounds may be, e.g., added to thereaction mixture, culture medium or injected into the cell.

If a sample containing a compound or a plurality of compounds isidentified in the method of the invention, then it is either possible toisolate the compound form the original sample identified as containingthe compound capable of acting as an agonist, or one can furthersubdivide the original sample, for example, if it consists of aplurality of different compounds, so as to reduce the number ofdifferent substances per sample and repeat the method with thesubdivisions of the original sample. Depending on the complexity of thesamples, the steps described above can be performed several times,preferably until the sample identified according to the method of theinvention only comprises a limited number of or only one substance(s).Preferably said sample comprises substances or similar chemical and/orphysical properties, and most preferably said substances are identical.Preferably, the compound identified according to the above-describedmethod or its derivative is further formulated in a form suitable forthe application in plant breeding or plant cell and tissue culture.

The term “early vigor” refers to the ability of a plant to grow rapidlyduring early is development, and relates to the successfulestablishment, after germination, of a well-developed root system and awell-developed photosynthetic apparatus.

The term “resistance to lodging” or “standability” refers to the abilityof a plant to fix itself to the soil. For plants with an erect orsemi-erect growth habit this term also refers to the ability to maintainan upright position under adverse (environmental) conditions. This traitrelates to the size, depth and morphology of the root system.

The term ‘grafting’ as used herein, refers to the joining together ofthe parts of two different plants so that they bind together and the sapcan flow, thus forming a single new plant that can grow and develop. Agraft therefore consists of two parts: (i) the lower part is therootstock as referred to herein and essentially consists of the rootsystem and a portion of the stem, and (ii) the upper part, the scion orgraft, which gives rise to the aerial parts of the plant.

As used herein, tblastn refers to an alignment tool that is part of theBLAST (Basic Local Alignment Search Tool) family of programs. BLAST aimsto identify regions of optimal local alignment, i.e., the alignment ofsome portion of two nucleic acid or protein sequences, to detectrelationships among sequences which share only isolated regions ofsimilarity (Altschul et al., 1990). In the present invention, tblastn ofthe BLAST 2.0 suite of programs was used to compare the maize cytokininoxidase protein sequence against a nucleotide sequence databasedynamically translated in all reading frames (Altschul et al., NucleicAcids Res. 25: 3389–3402 (1997)).

The following examples and figures are given by means of illustration ofthe present invention and are in no way limiting. The contents of allreferences included in this application are incorporated by reference.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1. Schematic representation of plant cytokinin oxidase genes.

Shown are the structures of different cytokinin oxidase genes isolatedfrom maize (ZmCKX1, accession number AF044603, Biochem. Biophys. Res.Corn. 255:328–333, 1999) and Arabidopsis (AtCKX1 to AtCKX4). Exons aredenominated with ‘E’ and represented by shaded boxes. Introns arerepresented by white boxes. Further indicated are the gene sizes (in kb,on top of each structure), the gene accession numbers (under the names)and a size bar representing 0.5 kb.

FIG. 2. Alignment of plant cytokinin oxidase amino acid sequences.

The amino acid sequences from cytokinin oxidases from maize (ZmCKX1) andArabidopsis (AtCKX1 to AtCKX4) are aligned. Identical amino acids aremarked by a black box, similar amino acid residues are in a grey box.Amino acid similarity groups: (M,I,L,V), (F, W, Y), (G, A), (S, T), (R,K, H,), (E, D), (N, Q). AtCKX1 is SEQ ID NO:2; AtCKX2 is SEQ ID NO:4;AtCKX3 is SEQ ID NO:6; AtCKX4 is SEQ ID NO:8.

FIG. 3. Northern blot analysis of AtCKX1-expressing tobacco andArabidopsis plants.

(A) Northern blot analysis of constitutively expressing tobacco plants(lanes 1–8) compared to wild type SNN tobacco (lane 9)

(B) Comparison of tetracycline-induced gene expression in leaves after12 h of induction with a constitutively expressing clone. Lanes 2–9,leaves of four different AtCKX1W38TetR clones (+,−, with or withouttetracycline treatment), lane 1, constitutively expressing 35S:: AtCKX1clone.

(C) Northern blot analysis of Arabidopsis plants constitutivelyexpressing AtCKX1 gene. Lanes 2–4, three different constitutivelyexpressing 35S::AtCKX1 clones compared to wild type Arabidopsis plant(lane 1).

FIG. 4: Growth characteristics of 35S::AtCKX1 transgenic Arabidopsisplants.

(A) Two wild type seedlings (left) compared to two 35S::AtCKX1expressing seedlings (right). Note the increased formation ofadventitious roots and increased root branching in the trangenicseedlings. Pictures were taken 14 days after germination. Plants weregrown in vitro on MS medium in petri dishes in a vertical position.

(B) Like A, but roots stained with toluidine blue.

(C) Top view of a petri dish with 35S::AtCKX1 transgenic seedlings threeweeks after germination.

(D) A 35S::AtCKX1 transgenic plants grown in liquid culture. Roots ofwild type seedlings grow poorly under these conditions (not shown).

(E) Transformants (T0) that express the 35S::AtCKX1 gene (three plantson the right), a wild type plant is shown on the left.

(F) Phenotype of T1 plants grown in soil. Wild type plant (left)compared to two 35S::AtCKX1 trangenic plants.

FIG. 5: Phenotype of AtCKX2 overexpressing Arabidopsis plants.

T1 generation of 35S::AtCKX2 expressing Arabidopsis plants (two plantson the right) compared to wild type (plant on the left).

FIG. 6. Northern blot analysis of AtCKX2 expressing tobacco andArabidopsis plants.

(A) Northern blot analysis of constitutively expressing tobacco plants(lanes 1–7) compared to wild type SNN tobacco (lane 8)

(B) Northern blot analysis of Arabidopsis plants constitutivelyexpressing AtCKX2 gene. Lanes 2–8, seven different consitutivelyexpressing 35S::AtCKX2 clones compared to wild type Arabidopsis plant(lane 1).

FIG. 7. Shoot phenotype of AtCKX1 and AtCKX2 expressing tobacco plants.

-   (A) Top view of six week old plants.-   (B) Tobacco plants at the flowering stage.-   (C) Kinetics of stem elongation. Arrows mark the onset of flowering.    Age of plants (days after germination) and leaf number at that stage    are indicated above the arrows. Bars indicate SD; n=12.-   (D) Number of leaves (n=12) formed between day 68 and day 100 after    germination and final surface area of these leaves (100% of wild    type is 3646±144 cm²; n=3).-   (E) Comparison of leaf size and senescence. Leaves were from nodes    number 4, 9, 12, 16 and 20 from the top (from left to right).

FIG. 8. Root phenotype of AtCKX expressing transgenic tobacco plants.

-   (A) Seedlings 17 days after germination.-   (B) Root system of soil grown plants at the flowering stage.-   (C) Root length, number of lateral roots (LR) and adventitious roots    (AR) on day 10 after germination.

(D) Dose-response curve of root growth Inhibition by exogenouscytokinin. Bars indicate±SD; n=30.

FIG. 9: Growth of axillary shoot meristems in 35S::AtCKX1 expressing,tobacco plants.

FIG. 10: Histology of shoot meristems, leaves and root meristems ofAtCKX1 overexpressing tobacco plants, versus wild type (WT) tobacco.

-   (A) Longitudinal median section through the vegetative shoot apical    meristem. P, leaf primordia.-   (B) Vascular tissue in second order veins of leaves. X, xylem, PH, a    phloem bundle.-   (C) Cross sections of fully developed leaves.-   (D) Scanning electron microscopy of the upper leaf epidermis.-   (E) Root apices stained with DAPI, RM, root meristem.-   (F) Longitudinal median sections of root meristems ten days after    germination. RC, root cap; PM, promeristem.-   (G) Transverse root sections 10 mm from the apex. E, epidermis,    C1–C4, cortical cell layer, X, xylem, PH, phloem. Bars are 100 μm.

FIG. 11: Northern blot analysis of AtCKX3 and AtCKX4-expressing tobaccoplants.

(A) Northern blot analysis of constitutively expressing AtCKX3 tobaccoplants. Lane designations indicate individual transgenic plant numbers,WT is wild type SNN tobacco. The blot on top was probed with a AtCKX3specific probe, the lower blot with a probe specific for the 25S rRNAand serves as a control for RNA loading.

(B) Northern blot analysis of constitutively expressing AtCKX4 tobaccoplants. Lane designations indicate individual transgenic plant numbers,WT is wild type SNN tobacco. The blot on top was probed with an AtCKX4specific probe, the lower blot with a probe specific for the 25S rRNAand serves as a control for RNA loading.

FIG. 12: Recipocal grafts of AtCKX transgenic tobacco plants and wildtype plants.

(A) Two plants on the left: Control (WT scion grafted on a WTrootstock).

Two plants on the right: WT scion grafted on a AtCKX2-38 transgenicrootstock.

(B) Left: Control (WT scion grafted on a WT rootstock).

Right: Scion of AtCKX2-38 plant grafted on WT rootstock.

(C) Magnification of root area.

Left: Control (WT scion grafted on a WT rootstock).

Right: WT scion grafted on an AtCKX2-38 transgenic rootstock.

(D) Formation of adventitious roots.

Left: Control (WT scion grafted on an WT rootstock).

Right: WT scion grafted on an AtCKX2-38 transgenic rootstock.

EXAMPLES Example 1 Brief Description of the Sequences of the Invention

Seq ID No Description 1 AtCKX1 genomic 2 AtCKX1 protein 3 AtCKX2 genomic4 AtCKX2 protein 5 AtCKX3 genomic 6 AtCKX3 protein 7 AtCKX4 genomic 8AtCKX4 protein 9 AtCKX5 genomic (short version) 10 AtCKX5 protein (shortversion) 11 AtCKX6 genomic 12 AtCKX6 protein 13 5′primer AtCKX1 143′primer AtCKX1 15 5′primer AtCKX2 16 3′primer AtCKX2 17 5′primer AtCKX318 3′primer AtCKX3 19 5′primer AtCKX4 20 3′primer AtCKX4 21 5′primerAtCKX5 22 3′primer AtCKX5 23 5′primer AtCKX6 24 3′primer AtCKX6 25AtCKX1 cDNA 26 AtCKX2 cDNA 27 AtCKX3 cDNA 28 AtCKX4 cDNA 29 AtCKX5 cDNA(short version) 30 AtCKX6 cDNA 31 AtCKX2 cDNA fragment 32 AtCKX2 peptidefragment 33 AtCKX5 genomic (long version) 34 AtCKX5 cDNA (long version)35 AtCKX5 protein (long version) 36 root clavata homolog promoter

Example 2 Identification of Candidate Cytokinin Oxidase Encoding Genesfrom Arabidopsis thaliana

Six different genes were identified from Arabidopsis thaliana that bearsequence similarity to a cytokinin oxidase gene from maize (Morris etal., Biochem Biophys Res Comm 255:328–333, 1999; Houda-Herin et al.Plant J 17:615–626; WO 99/06571). These genes were found by screening6-frame translations of nucleotide sequences from public genomicdatabases with the maize protein sequence, employing tblastn program.These sequences were designated as Arabidopsis thaliana cytokininoxidase-like genes or AtCKX. They were arbitrarily numbered as AtCKX1 toAtCKX6. The below list summarizes the information on these genes. Thepredicted ORF borders and protein sequences are indicative, in order toIllustrate by approximation the protein sequence divergence between theArabidopsis and maize cytokinin oxidases, as well as amongst thedifferent Arabidopsis cytokinin oxidases. The ORF borders and proteinsequences shown should not be taken as conclusive evidence for the modeof action of these AtCKX genes. For DNA and protein sequence comparisonsthe program MegAlign from DNAstar was used. This program uses theClustal method for alignments. For multiple alignments of protein andcDNA sequences the gap penalty and gap length penalty was set at 10each. For pairwise alignments of proteins the parameters were asfollows: Ktuple at 1; Gap penalty at 3; window at 5; diagonals saved at5. For pairwise alignments of cDNA's the parameters were as follows:Ktuple at 2; Gap penalty at 5; window at 4; diagonals saved at 4. Thesimilarity groups for protein alignments was: (M,I,L,V), (F,W,Y), (G,A),(S,T), (R,K,H), (E,D), (N,Q). The values that are indicated amongst theArabidopsis cDNA and protein sequences represent the lowest and highestvalues found with all combinations.

A. Gene name: AtCKX1 (Arabidopsis thaliana cytokinin oxidase-likeprotein 1, SEQ ID NO1)

Location in database (accession number, location on bac): AC002510,Arabidopsis thaliana chromosome II section 225 of 255 of the completesequence. Sequence from clones T32G6.

ORF Predicted in the Database:

15517 . . . 16183, 16415 . . . 16542, 16631 . . . 16891, 16995 . . .17257, 17344 . . . 17752

The AtCKX1 cDNA sequence is listed as SEQ ID NO 25

Predicted protein sequence: SEQ ID NO 2

Homologies

% Identity with Z. mays cDNA:

-   -   31.5% (Dnastar/MegAlign—Clustal method)        % Similarity with Z. mays Protein:    -   32.2% (Dnastar/MegAlign—Clustal method)        % Identity with Other Arabidopsis cDNA's (Range):    -   38.2% (AtCKX2)–54.1% (AtCKX6) (Dnastar/MegAlign—Clustal method)        % Similarity with Other Arabidopsis Proteins (Range):    -   37.1% (AtCKX2)–58.1% (AtCKX6) (Dnastar/MegAlign—Clustal method)

B. Gene name: AtCKX2 (Arabidopsis thaliana cytokinin oxidase-likeprotein 2, SEQ ID NO3)

Location in database (accession number, location on bac): AC005917,Arabidopsis thaliana chromosome II section 113 of 255 of the completesequence. Sequence from clones F27F23, F3P11.

ORF Predicted in the Database:

complement, 40721 . . . 41012, 41054 . . . 41364, 41513 . . . 41770,42535 . . . 42662, 43153 . . . 43711

Please note: The cDNA sequence identified by the inventor using the geneprediction program NetPlantGene was different than the one annotated inthe database. Based on the new cDNA sequence the ORF predicted in thedatabase was revised:

complement, 40721 . . . 41012, 41095 . . . 41364, 41513 . . . 41770,42535 . . . 42662, 43153 . . . 43711. The protein sequence encoded bythis cDNA is listed as SEQ. ID NO 4. The cDNA of AtCKX2 was cloned byRT-PCR from total RNA of AtCKA2 transgenic plant tissue with theone-step RT-PCR kit (Qiagen, Hilden, Germany) and sequenced using an ABIPRISM Big Dye Terminator cycle sequencing reaction kit (Perkin ElmerApplied Biosystems Division). This confirmed that the cDNA sequenceidentified and predicted by the inventor was correct. The new AtCKX2cDNA sequence is listed as SEQ ID NO 26. An 84-bp fragment correspondingto nucleotides 1171 through 1254 of the AtCKX2 cDNA is listed as SEQ IDNO 31. The corresponding peptide sequence of this 84-bp cDNA sequence islisted as SEQ ID NO 32.

Homologies

% Identity with Z. mays cDNA:

-   -   38.4% (Dnastar/MegAlign—Clustal method)        % Similarity with Z. mays Protein:    -   37.5% (Dnastar/MegAlign—Clustal method)        % Identity with Other Arabidopsis cDNA's (Range):    -   34.9% (AtCKX6)–64.5% (AtCKX4) (Dnastar/MegAlign—Clustal method)        % Similarity with Other Arabidopsis Proteins (Range):    -   36.5% (AtCKX6)–66.1% (AtCKX4) (Dnastar/MegAlign—Clustal method)

C. Gene name: AtCKX3 (Arabidopsis thaliana cytokinin oxidase-likeprotein 3, SEQ ID NO 5)

Location in database (accession number, location on bac): AB024035,Arabidopsis thaliana genomic DNA, chromosome 5, P1 clone: MHM17,complete sequence.

No prediction of the ORF in the database.

The gene was identified by the inventor using several gene predictionprograms including GRAIL, Genscan and NetPlantGene:

complement, 29415 . . . 29718, 29813 . . . 30081, 30183 . . . 30443,30529 . . . 30656, 32107 . . . 32716. The new AtCKX3 cDNA sequenceidentified by the inventor is listed as SEQ ID NO 27.

Predicted protein sequence, based on own ORF prediction: SEQ ID NO 6

Homologies

% Identity with Z. mays cDNA:

-   -   38.7% (Dnastar/MegAlign—Clustal method)        % Similarity with Z. mays Protein:    -   39.2% (Dnastar/MegAlign—Clustal method)        % Identity with Other Arabidopsis cDNA's (Range):    -   38.8% (AtCKX6)–51.0% (AtCKX2) (Dnastar/MegAlign—Clustal method)        % Similarity with Other Arabidopsis Proteins (Range):    -   39.9% (AtCKX6)–46.7% (AtCKX2) (Dnastar/MegAlign—Clustal method)

D. Gene name: AtCKX4 (Arabidopsis thaliana cytokinin oxidase-likeprotein 4, SEQ ID NO 7)

Location in database (accession number, location on bac):

-   1) AL079344, Arabidopsis thaliana DNA chromosome 4, BAC clone T16L4    (ESSA project)-   2) AL161575, Arabidopsis thaliana DNA chromosome 4, contig fragment    No. 71.    ORF Predicted in the Database:-   1) 76187 . . . 76814, 77189 . . . 77316, 77823 . . . 78080, 78318 .    . . 78586, 78677 . . . 78968-   2) 101002 . . . 101629, 102004 . . . 102131, 102638 . . . 102895,    103133 . . . 103401, 103492 . . . 103783

The AtCKX4 cDNA sequence is listed as SEQ ID NO 28

Predicted protein sequence: SEQ ID NO 8

Homologies

% Identity with Z mays cDNA:

-   -   41.0% (Dnastar/MegAlign—Clustal method)        % Similarity with Z. mays Protein:    -   41.0% (Dnastar/MegAlign—Clustal method)        % Identity with Other Arabidopsis cDNA's (Range):    -   35.2% (AtCKX6)–64.5% (AtCKX2) (Dnastar/MegAlign—Clustal method)        % Similarity with Other Arabidopsis Proteins (Range):    -   35.1% (AtCKX6)–66.1% (AtCKX2) (Dnastar/MegAlign—Clustal method)

E. Gene name: AtCKX5 (Arabidopsis thaliana cytokinin oxidase-likeprotein 5, SEQ ID NO 9)

Location in database (accession number, location on bac): AC023754,F1B16, complete sequence, chromosome 1

No prediction of the ORF in the database.

The gene was identified by the inventors using several gene predictionprograms including GRAIL, Genscan and NetPlantGene.

43756 . . . 44347, 44435 . . . 44562, 44700 . . . 44966, 45493 . . .45755, 46200 . . . 46560

The new AtCKX5 cDNA sequence identified and predicted by the inventor islisted as SEQ ID NO 29. The predicted protein sequence for this cDNA islisted as SEQ ID NO 10. A second potential ATG startcodon is present 9nucleotides more upstream in the genomic sequence. It is unclear whichof these 2 startcodons encodes the first amino acid of the protein.Therefore, a second potential AtCKX5 cDNA starting at this upstreamstartcodon is also listed in this invention as SEQ ID NO 34. Thecorresponding genomic sequence is listed as SEQ ID NO 33 and the encodedprotein as SEQ ID NO 35.

Homologies

% Identity with Z. mays cDNA:

-   -   39.1% (Dnastar/MegAlign—Clustal method)        % Similarity with Z. mays Protein:    -   36.6% (Dnastar/MegAlign—Clustal method)        % Identity with Other Arabidopsis cDNA's (Range):    -   40.1% (AtCKX2)–44.0% (AtCKX3) (Dnastar/MegAlign—Clustal method)        % Similarity with Other Arabidopsis Proteins (Range):    -   41.6% (AtCKX4)–46.4% (AtCKX6) (Dnastar/MegAlign—Clustal method)

F. Gene name: AtCKX6 (Arabidopsis thaliana cytokinin oxidase-likeprotein 6, SEQ ID NO 11)

Location in database (accession number, location on bac): AL163818,Arabidopsis thaliana DNA chromosome 3, P1 clone MA21 (ESSA project).

ORF Predicted in the Database:

46630 . . . 47215, 47343 . . . 47470, 47591 . . . 47806, 47899 . . .48161, 48244 . . . 48565

The AtCKX6 cDNA sequence is listed as SEQ ID NO 30

Predicted protein sequence: SEQ ID NO 12

Homologies

% Identity with Z. mays cDNA:

-   -   37.3% (Dnastar/MegAlign—Clustal method)        % Similarity with Z. mays Protein:    -   36.1% (Dnastar/MegAlign—Clustal method)        % Identity with Other Arabidopsis cDNA's (Range):    -   34.9% (AtCKX2)–54.1% (AtCKX1) (Dnastar/MegAlign—Clustal method)        % Similarity with Other Arabidopsis Proteins (Range):    -   35.1% (AtCKX4)–58.1% (AtCKX1) (Dnastar/MegAlign—Clustal method)

Genes AtCKX3 and AtCKX5 were not annotated as putative cytokininoxidases in the database and ORFs for these genes were not given.Furthermore, the ORF (and consequently the protein structures) predictedfor AtCKX2 was different from our own prediction and our prediction wasconfirmed by sequencing the AtCKX2 cDNA.

A comparison of the gene structure of the Arabidopsis AtCKX genes 1 to 4and the maize CKX gene is shown in FIG. 1.

The predicted proteins encoded by the Arabidopsis AtCKX genes showbetween 32% and 41% sequence similarity with the maize protein, whilethey show between 35% and 66% sequence similarity to each other. Becauseof this reduced sequence conservation, it is not clear a priori whetherthe Arabidopsis AtCKX genes encode proteins with cytokinin oxidaseactivity. An alignment of the Arabidopsis AtCKX predicted proteins 1 to4 and the maize CKX gene is shown in FIG. 2.

Example 3 Transgenic Plants Overexpressing AtCKX1 Showed IncreasedCytokinin Oxidase Activity and Altered Plant Morphology

1. Description of the Cloning Process

The following primers were used to PCR amplify the AtCKX1 gene fromArabidopsis thaliana, accession Columbia (non-homologous sequences usedfor cloning are in lower case):

-   Sequence of 5′ primer: cggtcgacATGGGATTGACCTCATCCTTACG (SEQ ID    NO:13)-   Sequence of 3′ primer: gcgtcgacTTATACAGTTCTAGGTTTCGGCAGTAT (SEQ ID    NO: 14)

A 2235-bp PCR fragment, amplified by these primers, was inserted in theSal I site of pUC19. The insert was sequenced and confirmed that the PCRamplification product did not contain any mutations. The SalI/SalIfragment of this vector was subcloned in the SalI site downstream of amodified CaMV 35S promoter (carrying three tetracycline operatorsequences) in the binary vector pBinHyg-Tx (Gatz et al., 1992). Theresulting construct was introduced into tobacco and Arabidopsis thalianathrough Agrobacterium-mediated transformation, using standardtransformation protocols.

2. Molecular Analysis of the Transgenic Lines

Several transgenic lines were identified that synthesize the AtCKX1transcript at high levels (FIG. 3). Transgenic lines expressing AtCKX1transcript also showed increased cytokinin oxidase activity asdetermined by a standard assay for cytokinin oxidase activity based onconversion of [2-³H]iP to adenine as described (Motyka et al., 1996).This is exemplified for 2 tobacco and 2 Arabidopsis lines in Table 6.This result proves that the AtCKX1 gene encodes a protein with cytokininoxidase activity.

TABLE 6 Cytokinin oxidase activity in AtCKX1 transgenic plant tissuesLeaf sample Cytokinin oxidase activity Plant species Plant line (nmolAde/mg protein.h) Arabidopsis Col-0 wild-type 0.009 CKX1-11 0.024CKX1-22 0.026 CKX1-22 0.027 Tobacco SNN wild-type 0.004 CKX1-SNN-8 0.016CKX1-SNN-28 0.0213. Phenotypic Description of the Transgenic Lines3.1 In Tobacco:

The plants had a dwarfed phenotype with reduced apical dominance (FIG.7A, B and C) and increased root production (FIG. 8).

Five Categories of Phenotype:

-   -   1) strong—2 clones    -   2) intermediate—3 clones    -   3) weak—4 clones    -   4) tall plants (as WT) with large inflorescence—5 clones    -   5) similar to WT, 9 clones

Height (see FIG. 7B and C)

-   -   WT: between 100–150 cm    -   weak: approximately 75 cm    -   intermediate: appr. 40–45 cm (main stem app. 25 cm but overgrown        by side branches.    -   strong: appr. 10 cm

The transgenics AtCKX1-48 and AtCKX1-50 displayed a strong phenotype.Below are measurements for stem elongation as compared to WT plants:

Line Wild-type AtCKX1-48 AtCKX1-50 Days after germination Height (cm)Height (cm) Height (cm) 47 9.5 ± 0.5 1.3 ± 0.3 1.2 ± 0.2 58 22.4 ± 2.3 2.2 ± 0.3 2.3 ± 0.3 68 35.3 ± 2.6  3.1 ± 0.5 2.6 ± 0.5 100 113.3 ± 9.8 7.1 ± 0.8 4.8 ± 0.9 117 138.6 ± 8.1  8.7 ± 0.7 6.6 ± 0.9 131 139.0 ±9.3  9.3 ± 0.7 8.6 ± 1.0 152 136.6 ± 10.4  10.9 ± 1.1  10.0 ± 1.0  16511.8 ± 1.9  11.4 ± 1.4  181 16.5 ± 1.7  14.9 ± 1.2  198 19.5 ± 1.5  18.1± 1.3 

-   -   Experimental: Plants were grown in soil in a greenhouse. Data        were collected from at least ten plants per line.        Leaves (see FIGS. 7D and E)

The shape of leaves of AtCKX1 transgenic expressors was lanceolate(longer and narrow): width-to-length ratio of mature leaves was reducedfrom 1:2 in wild type plants to 1:3 in AtCKX1 transgenics (FIG. 7E). Thenumber of leaves and leaf surface was reduced compared to WT (see FIG.7D). A prominent difference was also noted for progression of leafsenescence. In WT tobacco, leaf senescence starts in the most basalleaves and leads to a uniform reduction of leaf pigment (FIG. 7E). Bycontrast, ageing leaves of strongly expressing AtCKX1 plants stayedgreen along the leaf veins and turned yellow in the intercostal regions,indicating altered leaf senescence. The texture of older leaves was morerigid.

Roots

In vitro grown plants highly expressing the gene were easilydistinguishable from the WT by their ability to form more roots whichare thicker (stronger) (FIG. 8A), as well as by forming aerial rootsalong the stem.

The primary root was longer and the number of lateral and adventitiousroots was higher as illustrated in FIG. 8C for AtCKX1-50 overexpressingseedlings (see also Example 9).

The dose-response curve of root growth inhibition by exogenous cytokininshowed that roots of transgenic seedlings are more cytokinin resistantthan WT roots (FIG. 8D). The resistance of AtCKX1 transgenics to iPR wasless marked than for AtCKX2, which is consistent with the smallerchanges in iP-type cytokinins in the latter (see Table 10).

A large increase in root biomass was observed for adult plants grown insoil (see FIG. 8B for a plant grown in soil for 4 to 5 months) despitethe fact that growth of the aerial plant parts was highly reduced.

Internode Distance

-   -   intermediate phenotype: the 5^(th) internode below inflorescence        is about 2.5 cm long and 9^(th) internode was about 0.5 cm long        compared to 5 cm and 2 cm for the length of the 5^(th) and        9^(th) internode respectively, in WT plants.    -   strong phenotype: plant AtCKX1-50 The length of the 20^(th)        internode from the bottom measured at day 131 after germination        was 1.3±0.4 mm compared to 39.2±3.8 mm for WT        Apical Dominance and Branching

More side branches were formed indicating reduced apical dominancecompared to WT plants during vegetative growth (see FIG. 9). The sidebranches overgrew the main stem, reaching a height of 40–45 cm forintermediate AtCKX1 expressors. Even secondary branches appeared.However, the buds were not completely released from apical dominance,i.e. lateral shoots did not really continue to develop. The reducedapical dominance might be due to reduced auxin production by the smallershoot apical meristem (see Example 10).

Reproductive Development

The onset of flowering in AtCKX1 transgenics was delayed, the number offlowers and the seed yield per capsule was reduced. The size of flowerswas not altered in transgenic plants and the weight of the individualseeds was comparable to the weight of seeds from wild type plants. Datafor two representative AtCKX1 transgenics is summarized below:

A. Onset of flowering Line Wild-type AtCKX1-48 AtCKX1-50 Flowering time106.2 ± 3.3 193.3 ± 4.3 191.8 ± 3.8 (DAG)

-   -   Experimental: Data collected for at least ten plants per line.        The full elongation of the first flower was defined as onset of        flowering. DAG=days after germination.

B. Number of seed capsules per plant Line Wild-type AtCKX1-48 AtCKX1-50Number of 83.33 ± 5.13 2.00 ± 1.00 2.60 ± 1.67 capsules

-   -   Experimental: Number of seed capsules was determined at least        from 5 different plants. Please note that these plants were        grown under greenhouse conditions during winter time. This        affects negatively the number of flowers that are formed, in        particular in the transgenic clones. However, the general        picture that they form a reduced number of flowers is correct.        n.d., not determined

C. Seed yield/capsule (mg) Line Wild-type AtCKX1-48 AtCKX1-50Seed/capsule (mg) 87.41 ± 28.75 23.83 ± 13.36 61.8 ± 40.66

-   -   Experimental: Seed yield was determined for at least 12 seed        capsules. The size of seed capsules was very variable, hence the        large standard deviations. n.d., not determined

D. Weight of 100 seeds (mg) Line Wild-type AtCKX1-48 AtCKX1-50 Seedsweight (mg) 9.73 ± 0.44 10.70 ± 1.60 9.54 ± 0.94

-   -   Experimental: The seed biomass was determined as the weight of        100 seed from at least 5 different seed capsules. n.d., not        determined        3.2 In Arabidopsis    -   onset of germination was same as for WT    -   the total root system was enlarged and the number of side roots        and adventitious roots was enhanced (see FIGS. 4A through D)    -   the growth of aerial organs was reduced resulting in a dwarfed        phenotype (see FIGS. 4E and F) and the leaf biomass was reduced.        Leaf and flower formation is delayed.    -   the life cycle was longer compared to VVT and the seed yield was        lower compared to WT

The following morphometric data illustrate these phenotypes:

Root Development

Line Wild-type AtCKX1-11 AtCKX1-15 A. Total length of the root systemLength (mm) 32.5 76.5 68.4 B. Primary root length Length (mm) 32.3 ± 3.852.3 ± 4.8 39.9 ± 4.2 C. Lateral roots (LR) length Length (mm)  0.2 ±0.4 15.6 ± 11.0 10.4 ± 7.6 D. Adventitious roots length Length (mm) 0.03± 0.18  8.6 ± 8.5 19.1 ± 11.0 E. Number of lateral roots (LR) Number ofLR  0.3 ± 0.5 10.4 ± 5.4  2.6 ± 1.1 F. Number of adventitious roots (AR)Number of AR 0.03 ± 0.18  1.6 ± 1.1  2.6 ± 1.1

-   -   Experimental: Measurements were carried out on plants 8 days        after germination in vitro on MS medium. At least 17 plants per        line were scored.        Shoot Development

A. Leaf surface AtCKX1-11-7 AtCKX1-11-12 AtCKX1-15-1 T3 T3 T3 homozygoushomozygous homozygous Line Wild-type plants plants plants Leaf 21.16 ±1.73 2.28 ± 0.58 2.62 ± 0.28 1.66 ± 0.22 surface (cm²)

-   -   Experimental: Leaf surface area of main rosette leaves formed        after 30 days after germination was measured, 3 plants per clone        were analysed.        Reproductive Development    -   Onset of flowering

AtCKX1-11 AtCKX2-2 AtCKX2-5 T3 T2 T2 heterozygous heterozygousheterozygous Line Wild-type plants plants plants Flowering 43.6 ± 5.869.7 ± 9.4 51.2 ± 4.1 45.1 ± 6.9 time (DAG)

-   -   Experimental: Plants were grown under greenhouse condition. At        least 13 plants per clone were analysed. DAG=days after        germination

Conclusion: The analysis of AtCKX1 transgenic Arabidopsis plantsconfirmed largely the results obtained from tobacco and indicates thegeneral nature of the consequences of a reduced cytokinin content. Thetotal root system was enlarged (the total root length was increased app.110–140% in AtCKX1 transgenics), the shoot developed more slowly(retarded flowering) and the leaf biomass was reduced. The seed yieldwas lower in the transgenics as well (data not shown).

Example 4 Transgenic Plants Overexpressing AtCKX Showed IncreasedCytokinin Oxidase Activity and Altered Plant Morphology

1. Description of the Cloning Process

The following primers were used to PCR amplify the AtCKX2 gene fromArabidopsis thaliana, accession Columbia (non-homologous sequences usedfor cloning are in lower case):

Sequence of 5′ primer: gcggtaccAGAGAGAGAAACATAAACAAATGGC (SEQ ID NO:15)Sequence of 3′ primer: gcggtaccCAATTTTACTTCCACCAAAATGC (SEQ ID NO:16)

A 3104-bp PCR fragment, amplified by these primers, was inserted in theKpnI site of pUC19. The insert was sequenced to check that nodifferences to the published sequence were introduced by the PCRprocedure. The KpnI/KpnI fragment of this vector was subcloned in theKpnI site downstream of a modified CaMV 35S promoter (carrying threetetracycline operator sequences) in the binary vector pBinHyg-Tx (Gatzet al., 1992). The resulting construct was introduced into tobacco andArabidopsis thaliana through Agrobacterium-mediated transformation,using standard transformation protocols.

2. Molecular Analysis of the Transgenic Lines

Several transgenic lines were identified that synthesize the AtCKX2transcript at high levels (FIG. 6). Transgenic lines expressing AtCKX2transcript also showed increased cytokinin oxidase activity. This isexemplified for 2 tobacco and 3 Arabidopsis lines in Table 7. Thisresult proves that the AtCKX2 gene encodes a protein with cytokininoxidase activity.

TABLE 7 Cytokinin oxidase activity in AtCKX2 transgenic plant tissuesSample Plant species and Cytokinin oxidase activity tissue Plant line(nmol Ade/mg protein.h) Arabidopsis callus Col-0 wild-type 0.037 CKX2-150.351 CKX2-17 0.380 CKX2-55 0.265 Tobacco leaves SNN wild-type 0.009CKX2-SNN-18 0.091 CKX2-SNN-19 0.0913. Phenotypic Description of the Transgenic Lines3.1 In Tobacco (see FIGS. 7 to 10):

Three Categories of Phenotype:

-   -   1) strong—15 clones (similar to intermediate phenotype of        AtCKX1)    -   2) weak—6 clones    -   3) others—similar to WT plants, 7 clones        Aerial Plant Tarts

The observations concerning plant height, internode distance, branching,leaf form and yellowing were similar as for AtCKX1 transgenics with somegenerally minor quantitative differences in that the dwarfingcharacteristics were more severe in AtCKX1 transgenics than in AtCKX2trangenics (compare AtCKX1 plants with AtCKX2 plants in FIGS. 7A and B).This is illustrated below for stem elongation and internode distancemeasurements of clones with a strong phenotype AtCKX2-38 and AtCKX2-40:

Stem elongation Line Wild-type AtCKX2-38 AtCKX2-40 Days after HeightHeight Height germination (cm) (cm) (cm) 47  9.5 ± 0.5  2.4 ± 0.1  2.6 ±0.2 58  22.4 ± 2.3  5.5 ± 0.7  5.3 ± 0.5 68  35.3 ± 2.6  7.1 ± 0.8  7.0± 0.7 100 113.3 ± 9.8 15.5 ± 2.5 20.3 ± 6.4 117 138.6 ± 8.1 19.8 ± 3.829.5 ± 6.0 131 139.0 ± 9.3 26.5 ± 7.0 33.4 ± 5.8 152 136.6 ± 10.4 33.7 ±6.3 33.9 ± 6.4 165 36.2 ± 4.3

-   -   Experimental: Plants were grown in soil in a green house. Data        were collected from at least ten plants per line.

Internode distance Line Wild-type AtCKX2-38 Internode distance 39.2 ±3.8 7.2 ± 1.6 (mm)

-   -   Experimental: The length of the 20^(th) internode from the        bottom was measured at day 131 after germination.        Roots

In vitro plants highly expressing the gone were easily distinguishablefrom WT plants by ability to form more roots which are thicker(stronger) as well as by forming aerial roots along the stem.

The primary root was longer and the number of lateral and adventitiousroots was higher as illustrated in FIG. 8C for AtCKX2-38 overexpressingseedlings (see also Example 9).

The dose-response curve of root growth inhibition by exogenous cytokininshowed that roots of transgenic seedllings were more cytokinin resistantthan WT roots (FIG. 8D). The resistance of AtCKX1-28 transgenics to iPRwas less marked than for AtCKX2-38, which is consistent with the smallerchanges in iP-type cytokinins in the latter (see Table 10).

An increase in fresh and dry weight of the root biomass of TO lines ofAtCKX2 transgenic plants compared to WT was observed for plant grown insoil, as illustrated in the following table:

Line Wild-type AtCKX2 (T0) Fresh weight 45.2 ± 15.4 77.1 ± 21.3 (g) Dryweight  6.3 ± 1.9  8.6 ± 2.2 (g)

-   -   Experimental: Six WT plants and six independent To lines of        35S::AtCKX2 clone were grown on soil. After flowering the root        system was washed with water, the soil was removed as far as        possible and the fresh weight and dry weight was measured.    -   An increase in fresh and dry weight of the root biomass was also        observed for F1 progeny of AtCKX2 transgenics grown in        hydroponics as compared to WT, as illustrated in the following        table:

Line Wild-type AtCKX2-38 AtCKX2-40 Fresh weight ROOT 19.76 ± 6.79 33.38± 7.76 50.04 ± 15.59 (g) Dry weight ROOT  2.36 ± 0.43  2.61 ± 0.39  3.52± 1.06 (g) Fresh weight SHOOT 159.8 ± 44.53 33.66 ± 2.67 48.84 ± 11.83(g) Fresh weight  8.24 ± 0.63  1.04 ± 0.18  1.08 ± 0.51 SHOOT/ROOT ratio

-   -   Experimental: Soil grown plants were transferred 60 days after        germination to a hydroponic system (Hoagland's solution) and        grown for additional 60 days. The hydroponic solution was        aerated continuously and replaced by fresh solution every third        day.

In summary, transgenic plants grown in hydroponic solution formedapproximately 65–150% more root biomass (fresh weight) than wild typeplants. The increase in dry weight was 10–50%. This difference ispossibly in part due to the larger cell volume of the transgenics. Thisreduces the relative portion of cell walls, which forms the bulk of drymatter material. The shoot biomass was reduced to 20%-70% of wild typeshoots. The difference in fresh weight leads to a shift in theshoot/root ratio, which was approximately 8 in wild type butapproximately 1 in the transgenic clones.

Conclusion:

An increase in root growth and biomass was observed for AtCKX2transgenic seedlings and adult plants grown under different conditionscompared to WT controls despite the fact that growth of the aerial plantparts is reduced. Quantitative differences were observed betweendifferent transgenic plants: higher increases in root biomass wereobserved for the strongest expressing clones.

Reproductive Development

The onset of flowering in AtCKX2 transgenics was delayed, the number offlowers and the seed yield per capsule was reduced. These effects werevery similar to those observed in the AtCKX1 transgenic plants but theywere less prominent in the AtCKX2 transgenics, as indicated in thetables below. The size of flowers was not altered in transgenic plantsand the weight of the individual seeds was comparable to the weight ofseeds from wild type plants.

Onset of flowering AtCKX1- AtCKX1- AtCKX2- AtCKX2- Line Wild-type 48 5038 40 Flowering 106.2 ± 3.3 193.3 ± 191.8 ± 140.6 ± 121.9 ± time 4.3 3.86.5 9.8 (DAG)

-   -   Experimental: Data collected for at least ten plants per line.        The full elongation of the first flower was defined as onset of        flowering. DAG=days after germination.

B. Number of seed capsules per plant AtCKX1- AtCKX2- AtCKX2- LineWild-type AtCKX1-48 50 38 40 Number 83.33 ± 5.13 2.00 ± 1.00 2.60 ± 4.30± n.d. of 1.67 2.58 capsules

-   -   Experimental: Number of seed capsules was determined at least        from 5 different plants. Please note that these plants were        grown under green house conditions during winter time. This        affects negatively the number of flowers that are formed, in        particular in the transgenic clones. However, the general        picture that they form a reduced number of flowers is correct.        n.d., not determined

C. Seed yield/capsule (mg) AtCKX1- AtCKX1- AtCKX2- AtCKX2- LineWild-type 48 50 38 40 Seed/ 87.41 ± 28.75 23.83 ± 61.8 ± 46.98 ± n.d.capsule 13.36 40.66 29.30 (mg)

-   -   Experimental: Seed yield was determined for at least 12 seed        capsules. The size of seed capsules was very variable, hence the        large standard deviations. n.d., not determined

D. Weight of 100 seeds (mg) AtCKX1- AtCKX2- AtCKX2- Line Wild-typeAtCKX1-48 50 38 40 Seeds 9.73 ± 0.44 10.70 ± 9.54 ± 10.16 ± n.d. weight1.60 0.94 0.47 (mg)

-   -   Experimental: The seed biomass was determined as the weight of        100 seed from at least 5 different seed capsules. n.d., not        determined        3.2 In Arabidopsis:

The following morphometric data were obtained for AtCKX2 transgenics:

Root Development

Line Wild-type AtCKX2-2 AtCKX2-5 A. Total length of the root systemLength (mm) 32.5 50.6 48.5 B. Primary root length Length (mm) 32.3 ± 3.830.7 ± 4.8 31.6 ± 6.8 C. Lateral roots length Length (mm)  0.2 ± 0.4 5.5 ± 9.0 1.9 ± 2.5 D. Adventitious roots length Length (mm) 0.03 ±0.18 14.4 ± 10.2 14.9 ± 9.1 E. Number of lateral roots (LR) Number of LR 0.3 ± 0.5  2.9 ± 2.3 1.9 ± 1.0 F. Number of adventitious roots (AR)Number of AR 0.03 ± 0.18  1.8 ± 0.9 1.8 ± 1.0

-   -   Experimental: Measurements were carried out on plants 8 d.a.g.        In vitro on MS medium. At least 17 plants per line were scored.        Shoot Development

Leaf surface AtCKX2-2 AtCKX2-5 AtCKX2-9 T2 T2 T2 heterozygousheterozygous heterozygous Line Wild-type plants plants plants Leaf 21.16± 1.73 8.20 ± 2.35 8.22 ± 0.55 7.72 ± 0.85 surface (cm²)

-   -   Experimental: Leaf surface area of main rosette leaves formed        after 30 days after germination was measured. 3 plants per clone        were analysed.        Reproductive Development

Onset of flowering AtCKX1-11 AtCKX2-2 AtCKX2-5 T3 T2 T2 heterozygousheterozygous heterozygous Line Wild-type plants plants plants Flowering43.6 ± 5.8 69.7 ± 9.4 51.2 ± 4.1 45.1 ± 6.9 time (DAG)

-   -   Experimental: Plants were grown under greenhouse condition. At        least 13 plants per clone were analysed. DAG=days after        germination.

Conclusion: Arabidopsis AtCKX2 transgenics had reduced leaf biomass anda dwarfing phenotype similar to AtCKX1 transgenics (compare FIG. 5 withFIG. 4F). The total root system was also enlarged in AtCKX2 transgenicArabidopsis. The total root length is increased approximately 50% inAtCKX2 transgenics. The AtCKX1 transgenics have longer primary roots,more side roots and form more adventitious roots. AtCKX2 transgenicslack the enhanced growth of the primary root but form more side rootsand lateral roots than WT.

Summary:

The phenotypes observed for AtCKX2 transgenics were very similar but notidentical to the AtCKX1 transgenics, which in turn were very similar butnot identical to the results obtained for the tobacco trangenics. Thisconfirms the general nature of the consequences of a reduced cytokinincontent in these two plant species and therefore, similar phenotypes canbe expected in other plant species as well. The main difference betweentobacco and Arabidopsis is the lack of enhanced primary root growth inAtCKX2 overexpressing plants (data not shown).

Example 5 Transgenic Plants Overexpressing AtCKX3 Showed IncreasedCytokinin Oxidase Activity and Altered Plant Morphology

1. Description of the Cloning Process

The following primers were used to PCR amplify the AtCKX3 gene fromArabidopsis thaliana, accession Columbia (non-homologous sequences usedfor cloning are in lower case):

-   Sequence of 5′ primer gcggtaccTTCATTGATAAGAATCMGCTATTCA (SEQ ID    NO:17)-   Sequence of 3′ primer: gcggtaccCAAAGTGGTGAGAACGACTAACA (SEQ ID    NO:18)

A 3397-bp PCR fragment, produced by this PCR amplification, was insertedin the KpnI site of pBluescript. The insert was sequenced to confirmthat the PCR product has no sequence changes as compared to the gene.The KpnI/KpnI fragment of this vector was subcloned in the KpnI sitedownstream of a modified CaMV 35S promoter (carrying three tetracyclineoperator sequences) in the binary vector pBinHyg-Tx (Gatz et al., 1992).The resulting construct was introduced into tobacco and Arabidopsisthaliana through Agrobacterium-mediated transformation, using standardtransformation protocols.

2. Molecular Analysis of the Transgenic Lines

Several transgenic tobacco lines were identified that synthesize theAtCKX3 transcript at high levels (FIG. 11A.). Transgenic tobacco linesexpressing AtCKX3 transcript also showed increased cytokinin oxidaseactivity. This is exemplified for three plants in Table 8. This provesthat the AtCKX3 gene encodes a protein with cytokinin oxidase activity.

TABLE 8 Cytokinin oxidase activity in AtCKX4 transgenic plant tissuesSample Plant species and Cytokinin oxidase activity tissue Plant line(nmol Ade/mg protein.h) tobacco leaves SNN wild-type 0.011 CKX3-SNN-30.049 CKX3-SNN-6 0.053 CKX3-SNN-21 0.053. Plant Phenotypic Analysis

The phenotypes generated by overexpression of the AtCKX3 gene in tobaccoand Arabidopsis were basically similar as those of AtCKX1 and AtCKX2expressing plants, i.e. enhanced rooting and dwarfing. However,overexpression of the AtCKX3 gene in tobacco resulted in a strongerphenotype compared to AtCKX2. In this sense AtCKX3 overexpression wasmore similar to AtCKX1 overexpression.

Example 6 Transgenic Plants Overexpressing AtCKX4 Showed IncreasedCytokinin Oxidase Activity and Altered Plant Morphology

1. Description of the Cloning Process

The following primers were used to PCR amplify the AtCKX4 gene fromArabidopsis thaliana, accession Columbia (non-homologous sequences usedfor cloning are in lower case):

-   Sequence of 5′ primer: gcggtaccCCCATTAACCTACCCGTTTG (SEQ ID NO:19)-   Sequence of 3′ primer: gcggtaccAGACGATGAACGTACTTGTCTGTA (SEQ ID    NO:20)

A 2890-bp PCR fragment, produced by this PCR amplification, was insertedin the KpnI site of pBluescript. The Insert was sequenced to confirmthat the PCR product has no sequence changes as compared to the gene.The KpnI/KpnI fragment of this vector was subcloned in the KpnI sitedownstream of a modified CaMV 35S promoter (carrying three tetracyclineoperator sequences) in the binary vector pBinHyg-Tx (Gatz et al., 1992).The resulting construct was introduced into tobacco and Arabidopsisthaliana through Agrobacterium-mediated transformation, using standardtransformation protocols.

2. Molecular Analysis of the Transgenic Lines

Several transgenic tobacco lines synthesized the AtCKX4 transcript athigh levels (FIG. 11B.). Transgenic lines expressing AtCKX4 transcriptalso showed increased cytokinin oxidase activity. This is exemplifiedfor 3 Arabidopsis and 3 tobacco lines in Table 9. This result provesthat the AtCKX4 gene encodes a protein with cytokinin oxidase activity.

TABLE 9 Cytokinin oxidase activity in AtCKX4 transgenic plant tissuesSample Plant species and Cytokinin oxidase activity tissue Plant line(nmol Ade/mg protein.h) Arabidopsis callus Col-0 wild-type 0.037 CKX4-370.244 CKX4-40 0.258 CKX4-41 0.320 tobacco leaves SNN wild-type 0.011CKX4-SNN-3 0.089 CKX4-SNN-18 0.085 CKX4-SNN-27 0.096

Overall, the data showed that the apparent K_(m) values for the fourcytokinin oxidases were in the range of 0.2 to 9.5 μM with IP assubstrate, which further demonstrates that the proteins encoded byAtCKX1 through 4 are indeed cytokinin oxidase enzymes as disclosedherein.

3. Plant Phenotypic Analysis

The phenotypes generated by overexpression of the AtCKX4 gene in tobaccoand Arabidopsis were basically similar as those of AtCKX1 and AtCKX2expressing plants, i.e. enhanced rooting, reduced apical dominance,dwarfing and yellowing of intercostal regions in older leaves oftobacco. An additional phenotype in tobacco was lanceolate leaves(altered length-to-width ratio).

General Observations of AtCKX Overexpressing Tobacco Plants

Overall, the phenotypic analysis demonstrated that AtCKX geneoverexpression caused drastic developmental alterations in the plantshoot and root system in tobacco, including enhanced development of theroot system and dwarfing of the aerial plant part. Other effects such asaltered leaf senescence, formation of adventitious root on stems, andothers were also observed as disclosed herein. The alterations were verysimilar, but not identical, for the different genes. In tobacco, AtCKX1and AtCKX3 overexpressors were alike as were AtCKX2 and AtCKX4.Generally, the two former showed higher expression of the traits,particularly in the shoot. Therefore, a particular cytokinin oxidasegene may be preferred for achieving the phenotypes that are described inthe embodiments of this invention.

Example 7 Cloning of the AtCKX5 Gene

The following primers were used to PCR amplify the AtCKX5 gene fromArabidopsis thaliana, accession Columbia (non-homologous sequences usedfor cloning are in lower case):

Sequence of 5′ primer: ggggtaccTTGATGAATCGTGAAATGAC (SEQ ID NO:21)Sequence of 5′ primer: ggggtaccCTTTCCTCTTGGTTTTGTCCTGT (SEQ ID NO:22)

The sequence of the 5′ primer includes the two potential startcodons ofthe AtCKX5 protein, the most 5′ startcodon is underlined and a secondATG is indicated in italics. A 2843-bp PCR fragment, produced by thisPCR amplification, was inserted as a blunt-end product in pCR-BluntII-TOPO cloning vector (Invitrogen).

Example 8 Cloning of the AtCKX6 Gene

The following primers were used to PCR amplify the AtCKX6 gene fromArabidopsis thaliana, accession Columbia (non-homologous sequences usedfor cloning are in lower case):

Sequence of 5′ primer: gctctagaTCAGGAAAAGAACCATGCTTATAG (SEQ ID NO:23)Sequence of 3′ primer: gctctagaTCATGAGTATGAGACTGCCTTTTG (SEQ ID NO:24)

A 1949-bp PCR fragment, produced by this PCR amplification, was insertedas a blunt-end product in pCR-Blunt II-TOPO cloning vector (Invitrogen).

Example 9 Tobacco Seedling Growth Test Demonstrated Early Vigor of AtCKXTransgenics

Seeds of AtCKX1-50 and AtCKX2-38 overexpressing transgenics and WTtobacco were sown in vitro on MS medium, brought to culture room 4 daysafter cold treatment and germinated after 6 days. Observations onseedling growth were made 10 days after germination (see also FIG. 8C)and are summarized below. At least 20 individuals were scored per clone.Similar data have been obtained in two other experiments.

Line Wild-type AtCKX1-50 AtCKX2-38 A. Total length of the root systemLength 61.1 122.0 106.5 (mm) B. Primary root length Length 32.3 ± 2.650.8 ± 4.5 52.4 ± 4.8 (mm) C. Lateral roots length Length  9.8 ± 5.518.0 ± 8.1 13.0 ± 6.0 (mm) D. Adventitious roots length Length 19.0 ±5.0 53.0 ± 12.0 42.0 ± 9.8 (mm) E. Number of lateral roots (LR) Numberof LR  1.9 ± 0.9  6.5 ± 2.2  5.6 ± 2.0 F. Number of adventitious roots(AR) Number of AR  2.2 ± 0.6  3.5 ± 0.9  3.6 ± 1.3AtCKX1 and AtCKX2 Plants, General Observations:

Seedlings of AtCKX1 and AtCKX2 overexpressing tobacco plants had 60%more adventitious roots and three times more lateral roots thanuntransformed control plants 10 days after germination. The length ofthe primary root was increased by about 70%. This—together with more andlonger side roots and secondary roots—resulted in a 70–100% increase intotal root length. These results showed that overexpression of cytokininoxidase enhances the growth and development of both the main root andthe adventitious roots, resulting in early vigor.

Example 10 Histological Analysis of Altered Plant Morphology in AtCKX1Overexpressing Tobacco Plants

Microscopic analysis of different tissues revealed that themorphological changes in AtCKX transgenics are reflected by distinctchanges in cell number and rate of cell formation (see FIG. 10). Theshoot apical meristem (SAM) of AtCKX1 transgenics was smaller than inwild type and fewer cells occupy the space between the central zone andthe peripheral zone of lateral organ fromation, but the cells were ofthe same size (FIG. 10A). The reduced cell number and size of the SAM asa consequence of a reduced cytokinin content indicates that cytokininshave a role in the control of SAM proliferation. No obvious changes inthe differentiation pattern occurred, suggesting that the spatialorganization of the differentiation zones in the SAM is largelyindependent from cell number and from the local cytokinin concentration.The overall tissue pattern of leaves in cytokinin oxidase overexpresserswas unchanged. However, the size of the phloem and xylem wassignificantly reduced (FIG. 10B). By contrast, the average cell size ofleaf parenchyma and epidermal cells was increased four- to fivefold(FIGS. 10C, D). New cells of AtCKX1 transgenics are formed at 3–4% ofthe rate of wild type leaves and final leaf cell number was estimated tobe in the range of 5–6% of wild type. This indicates an absoluterequirement for cytokinins in leaves to maintain the cell divisioncycle. Neither cell size nor cell form of floral organs was altered andseed yield per capsule was similar in wild type and AtCKX transgenicplants. The cell population of root meristems of AtCKX1 transgenicplants was enlarged approximately 4-fold and the cell numbers in boththe central and lateral columnella were enhanced (FIGS. 10E, F). Thefinal root diameter was increased by 60% due to an increased diameter ofall types of root cells. The radial root patterns was Identical in wildtype and transgenics, with the exception that frequently a fourth layerof cortex cells was noted in transgenic roots (FIG. 10G). The increasedcell number and the slightly reduced cell length indicates that theenhanced root growth is due to an increased number of cycling cellsrather than increased cell growth. In the presence of lowered cytokinincontent, root meristem cells must undergo additional rounds of mitosisbefore they leave the meristem and start to elongate. The exit from themeristem is therefore regulated by a mechanism that is sensitive tocytokinins. Apparently, cytokinins have a negative regulatory role inthe root meristem and wild type cytokinin concentrations are inhibitoryto the development of a maximal root system. Therefore, reducing thelevel of active cytokinins by overexpressing cytokinin oxidasesstimulates root development, which results in an increase in the size ofthe root with more lateral and adventitious roots as compared to WTplants.

Example 11 AtCKX1 and AtCKX2-Overexpressing Tobacco Plants had a ReducedCytokinin Content

Among the 16 different cytokinin metabolites that were measured, thegreatest change occurred in the iP-type cytokinins in AtCKX2overexpressers (Table 10): the overall decrease in the content ofiP-type cytokinins is more pronounced in AtCKX2 expressing plants thanin AtCKX1 transgenics. AtCKX1 transgenics showed a stronger phenotype inthe shoot. It is not known which cytokinin metabolite is relevant forthe different traits that were analyzed. It may be that differentcytokinin forms play different roles in the various developmentprocesses. Smaller alterations were noted for Z-type cytokinins, whichcould be due to a different accessibility of the substrate or a lowersubstrate specificity of the protein. The total content of iP and Zmetabolites in individual transgenic clones was between 31% and 63% ofwild type. The cytokinin reserve pool of O-glucosides was also loweredin the transgenics (Table 10). The concentration of N-glucosides andDHZ-type cytokinins was very low and was not or only marginally, alteredin transgenic seedlings (data not shown).

TABLE 10 Cytokinin content of AtCKX transgenic plants. Cytokininextraction, immunopurification, HPLC separation and quantification byELISA methods was carried out as described by Faiss et al., 1997. Threeindependently pooled samples of approximately 100 two week old seedlings(2.5 g per sample) were analyzed for each clone. Concentrations are inpmol × g fresh weight⁻¹. Abbreviations: iP, N⁶- (Δ²isopentenyl)adenine;iPR, N⁶-(Δ²isopentenyl)adenine riboside; iPRP, N⁶-(Δ²isopentenyl)adenine riboside 5′-monophosphate; Z, trans-zeatin; ZR,zeatin riboside; ZRP, zeatin riboside 5′-monophosphate; ZOG, zeatinO-glucoside; ZROG, zeatin riboside O-glucoside. Line AtCKX1-2 AtCKX1-28AtCKX2-38 AtCKX2-40 Cytokinin WT % of % of % of % of metaboliteConcentration Concentration WT Concentration WT Concentration WTConcentration WT IP 5.90 ± 1.80 4.76 ± 0.82 81 4.94 ± 2.62 84 1.82 ±0.44 31 2.85 ± 0.62 48 IPR 2.36 ± 0.74 1.53 ± 0.14 65 0.75 ± 0.27 320.55 ± 0.39 23 0.89 ± 0.07 38 IPRP 3.32 ± 0.73 0.87 ± 0.26 26 1.12 ±0.13 34 0.80 ± 0.48 24 1.68 ± 0.45 51 Z 0.24 ± 0.06 0.17 ± 0.02 71 0.22± 0.03 92 0.21 ± 0.06 88 0.22 ± 0.02 92 ZR 0.60 ± 0.13 0.32 ± 0.12 530.34 ± 0.03 57 0.34 ± 0.15 57 0.32 ± 0.05 53 ZRP 0.39 ± 0.17 0.42 ± 0.11107 0.28 ± 0.15 72 0.06 ± 0.01 15 0.17 ± 0.06 44 ZOG 0.46 ± 0.20 0.32 ±0.09 70 0.26 ± 0.13 57 0.20 ± 0.07 43 0.12 ± 0.02 26 ZROG 0.48 ± 0.170.30 ± 0.06 63 0.47 ± 0.02 98 0.23 ± 0.05 48 0.30 ± 0.13 63 Total 13.758.69 63 8.38 61 4.21 31 6.55 48

Example 12 Grafting Experiments Showed that Dwarfing and Enhanced RootDevelopment due to AtCKX Overexpression is Confined to TransgenicTissues

To investigate which phenotypic effects of cytokinin oxidaseoverexpression are restricted to expressing tissues, i.e. are cell- ororgan-autonmous traits, grafting experiments were performed. Reciprocalgrafts were made between an AtCKX2 transgenic tobacco plant and a WTtobacco. The transgenic plant used in this experiment was AtCKX2-38,which displayed a strong phenotype characterized by enhanced root growthand reduced development of the aerial plant parts. As described inExample 3 through 6, these were two important phenotypes that resultedfrom cytokinin oxidase overexpression in tobacco and arabidopsis.

Plants were about 15 cm tall when grafted and the graft junction wasabout 10 cm above the soil. FIG. 12 shows plants 15 weeks aftergrafting. The main results were that: (i) the aerial phenotype of a WTscion grafted on a transgenic rootstock was similar to the WT controlgraft (=WT scion on WT rootstock). Importantly, this showed thatoverexpression of the AtCKX2 transgene in the rootstock did not inducedwarfing of the non-transgenic aerial parts of the plant (see FIG. 12A).Improved root growth of the transgenic rootstock was maintained,indicating that improved root growth of AtCKX transgenics is autonomousand does not depend on an AtCKX transgenic shoot (FIG. 12C).Interestingly, the WT scions grafted on the transgenic rootstocks lookedhealthier and were better developed. Notably, senescence of the basalleaves was retarded in these plants (see FIG. 12A); (ii) the transgenicscion grafted on the WT rootstock looked similar to the aerial part ofthe transgenic plant from which it was derived, i.e. the shoot dwarfingphenotype is also autonomous and not dependent on the improved rootgrowth (see FIG. 12B).

In addition to the above-mentioned better appearance of WT shootsgrafted on a transgenic rootstock, the formation of adventitious rootson the basal part of WT shoots was noted (FIG. 12D, right plant).Formation of adventitious roots also occurred on the stem of AtCKXtransgenics but not on stems of WT control grafts (FIG. 12D, left plant)and therefore seems to be a non-autonomous trait.

In summary, it is disclosed in this invention that enhanced rootformation and dwarfing of the shoot in AtCKX overexpressing tobacco areautonomous traits and can be uncoupled by grafting procedures.Surprisingly, grafting of a WT scion on an AtCKX transgenic rootstockresulted in more vigourosly growing plants and retardation of leafsenescence.

As an alternative to grafting, tissue-specific promoters could be usedfor uncoupling the autonomous phenotypic effects of cytokininoverexpression. Therefore, it is disclosed in this invention thatcytokinin oxidase overexpression in a tissue specific manner can be usedto alter the morphology of a plant such as the shoot or root system.

Example 13 Expression of an AtCKX Gene Under a Root-Specific Promoter inTransgenic Plants Leads to Increased Root Production

An AtCKX gene (see example 4) is cloned under control of the rootclavata homolog promoter of Arabidopsis (SEQ ID NO 36), which is apromoter that drives root-specific expression. Other root-specificpromoters may also be used for the purpose of this invention. See Table5 for exemplary root-specific promoters.

Transgenic plants expressing the AtCKX gene specifically in the rootsshow increased root production without negatively affecting growth anddevelopment of the aerial parts of the plant. Positive effects on leafsenescence and growth of aerial plant parts are observed.

Example 14 Suppression of an AtCKX Gene Under a Senescence-InducedPromoter in Transgenic Plants Leads to Delayed Leaf Senescence andEnhanced Seed Yield

A chimeric gene construct derived from an AtCKX gene and designed tosuppress expression of endogenous cytokinin oxidase gene(s) is clonedunder control of a senescence-induced promoter. For example, promotersderived from senescence-associated genes (SAG) such as the SAG12promoter can be used (Quirino et al., 2000). Transgenic plantssuppressing endogenous cytokinin oxidase gene(s) specifically insenescing leaves show delayed leaf senescence and higher seed yieldwithout negatively affecting the morphology and growth and developmentof the plant.

Example 15 Overexpression of an AtCKX Gene in the Female ReproductiveOrgans Leads to Parthenocarpic Fruit Development

The open reading frame of an AtCKX gene is cloned under control of apromoter that confers overexpression in the female reproductive organssuch as for example the DefH9 promoter from Antirrhinum majus or one ofits homologues, which have high expression specificity in the placentaand ovules. Transgenic plants with enhanced cytokinin oxidase activityin these tissues show parthenocarpic fruit development.

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1. An isolated polypeptide comprising the amino acid sequence of SEQ IDNO:4.
 2. The isolated polypeptide of claim 1, wherein at least one of abinding or activation domain of a transcriptional activator, phage coatprotein, (histidine)6-tag, glutathione S-transferase, protein A,maltose-binding protein, dihydrofolate reductase, Tag. 100 epitope (SEQID NO:37), c-myc epitope (SEQ ID NO:38), Flag®-epitope (SEQ ID NO:39),lacZ, calmodulin-binding peptide (CMP), HA epitope (SEQ ID NO:40),protein C epitope (SEQ ID NO:41) or VSV epitope (SEQ ID NO:42) is fusedto the amino- or carboxy-terminus of said polypeptide and thepolpypeptide continues to have cytokinin oxidase activity.